Factors affecting the attraction of Diptera towards human carcasses - a literature study

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Factors affecting the attraction of Diptera

towards human carcasses – a literature study

Masterthesis BSc. L.O.N. Buijs

Student number: 12697907 Master Forensic Science

University of Amsterdam (UvA) Number of words: 12.167

Supervisor: Dhr. Dr. J.A.J. Breeuwer Examiner: Dhr. Dr. P. Roessingh 29th_{of November 2020}

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Abstract

Forensic entomology, the study of insects associated with homicides and abuse cases, is used during criminal investigations to estimate the time since death or so-called post-mortem interval (PMI). The PMI can be of great importance for the identification of victims or perpetrators. In forensic entomology insects present on a body are used to estimate the time of death. However, currently the pre-colonization interval is not taken into account when doing PMI estimations. This thesis aims to identify factors affecting the detection of human cadavers by flies (Diptera) and thereby the pre-colonization interval. First, the use of forensic entomology in PMI estimations, the production of volatile compounds during human decomposition and the detection of these compounds by Diptera are discussed. The results suggest that sulphide-containing compounds, produced during protein breakdown, play a role in detection of carcasses by Diptera. Then, various factors that may accelerate or inhibit the detection and attraction of Diptera towards human cadavers are discussed. The effect of physical barriers, burning, chemical modifications and intrinsic factors on Diptera attraction are investigated. The results show that especially the hiding and burial of bodies, as well as the treatment of bodies with chemical substances affect the attraction of Diptera towards human carcasses. Intrinsic factors, such as the antemortem use of drugs, alcohol and medication of the bodies, are hardly studied today. This literature study gives an overview of forensic entomology and links biological information about insects to the decay of carcasses and the impact on PMI estimations. At the same time it is clear that more harmonisation in research is needed in order to get more insights into the interaction between flies and human cadavers and the pre-colonization interval.

Introduction

Forensic entomology is the branch of forensic science that focusses on the study of insects associated with criminal investigations such as homicides, abuse and animal cruelty cases (Amendt et al., 2007; I. Joseph et al., 2011). Focusing on dead persons, insects and other arthropods are attracted to decomposing bodies and will try to oviposit/larviposit (lay eggs/larvae) in it (Rivers & Dahlem, 2014; Smith, 1986). Studying the distribution, biology and behaviour of these insects can provide information about multiple aspects, such as the cause of death, movement of the corpse and most importantly the post-mortem interval (PMI). The PMI is the time elapsed since the death of a person (Amendt et al., 2007; I. Joseph et al., 2011). Before insects arrive at a body and leave physical evidence (such as larvae) behind, the corpse first has to be detected and found by the insects. This so-called precolonization interval is part of the PMI, but cannot be estimated using the insects present when the body is found (Tomberlin et al., 2011). It is therefore important to know what factors influence the detection and attraction of insects towards corpses. Especially as bodies in criminal cases are often hidden or modified in order to prevent discovery (Tomberlin et al., 2011). In this thesis I will look into the following research question: What factors affect the attraction of Diptera to human cadavers?

In order to answer this research question, it is needed to have knowledge about the compounds that are produced by cadavers, how these volatiles can be detected by flies and how flies are subsequently attracted towards cadavers. This thesis is divided into five chapters, starting with a general chapter introducing the use of insect succession in PMI estimations [chapter 1]. Next, the way in which insects perceive and process their environment is discussed [chapter 2], knowledge needed for the subsequent discussion of the decomposition volatiles that can be detected by Diptera [chapter 3 and 4]. I will focus on insects of the order Diptera (flies) as these are the main insects involved in early cadaver detection and colonization (I. Joseph et al., 2011). Finally, various factors affecting the detection of human cadavers by flies will be discussed [chapter 5]. In each chapter the results are discussed. In the concluding chapter, the most important findings and recommendations for future research will be given.

Chapter 1: Insect succession and PMI estimations

In general four categories of insects are found on decomposing carrion: [1] necrophagous species feeding on the carrion, such as Calliphoridae (blow flies) and Coleoptera (beetles); [2] predators and parasites feeding on the necrophagous species; [3] omnivorous species feeding on the carrion and other arthropods; [4] other species like spiders that use the cadaver as a part of their environment (Smith, 1986). The first group is the most important in estimating the PMI. The succession of the insects and other organisms associated with decomposition is dependent on the changes taking place in a corpse after death (Smith, 1986). At the start of decomposition, the cadaver will be detected by arthropods and subsequently colonized. As the decomposition of the body proceeds, waves of insect colonization will proceed in a sequential manner (Rivers & Dahlem, 2014; Smith, 1986). This is called entomofaunal succession (Frederickx et al., 2012). However, carrion decomposition and insect succession are continuous processes, with different waves of insects overlapping or returning in different stages of

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4 decomposition. Decomposition, and the associated succession of insects, is dependent on all kinds of biotic and abiotic factors, making the relationship between necrophagous insects and a corpse highly unpredictable (Rivers & Dahlem, 2014).

In forensic investigations the order Diptera (true flies) is the most important. The main species in this order are the families Calliphoridae (blow flies) and Sacrophagidae (flesh flies) which can arrive within minutes after death and form the first wave of insect colonization on the body (I. Joseph et al., 2011). In particular their larval activities contribute further to the process of decomposition and disintegration of the body (Smith, 1986). In addition, the presence of these insects and the chemicals excreted by them can affect the attraction of other insects towards the cadaver (Rivers & Dahlem, 2014)

1.1 PMI estimation

Insects can provide information on several forensic topics, but the main focus lies on the information about corpses. As the growth and development of necrophagous species are correlated to feeding on the body and ambient temperatures, these insects can be used to estimate the time since death (PMI). When a human corpse is discovered, the investigation focuses on determining the time and causation of death. This process is very complex, as decomposition is affected by many factors, such as location, humidity, temperature and physical appearance of the body itself (A. Vass, 2001). Forensic entomology can contribute to the estimation of the PMI. However, it is an estimate; the exact moment of death cannot be determined (Goff, 2009; Rivers & Dahlem, 2014).

Early after death multiple methods associated with forensic medicine, such as measurement of the body temperature, provide a fairly accurate picture of the onset of death, but after 48-72 hours the estimation of the PMI with these techniques becomes less accurate (Goff, 2009; Rivers & Dahlem, 2014). After 72 hours, forensic entomology can be used to contribute to PMI estimations (Rivers & Dahlem, 2014). The PMI is very important in criminal investigations, as it can help to find the person responsible for the death of an individual and the identification of the victim. Also the PMI can be of main importance in cases of neglect and abuse (Rivers & Dahlem, 2014).

Currently, pretty accurate estimations can be made of the interval between the first arrival of arthropods and the discovery and investigation of the body, the so-called postcolonization interval (post-CI) or period of insect activity (PIA) (Rivers & Dahlem, 2014; Tomberlin et al., 2011). The PMI resulting from estimating the post-CI is defined as the PMImin, which corresponds to the time between insect colonization and discovery of the body (Rivers & Dahlem, 2014). However, a problem arises when estimations are made of the time it takes for insects to detect a body, the so-called precolonization interval (pre-CI) (Tomberlin et al., 2011). During insect colonization, the insects leave evidence of carrion feeding and oviposit/larviposit on the body, which can be studied by forensic entomologists (I. Joseph et al., 2011; Tomberlin et al., 2011). For the estimation of the post-CI often the growth and development rate of the larvae present on the body are used. By species identification and measurements of mass and length of the larvae, the age of the larvae can be estimated (I. Joseph et al., 2011; Tomberlin et al., 2011). As the development of insect larvae is largely dependent on temperature, temperature

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5 information of the days before discovery are used in the estimation of the age of the larvae (Tomberlin et al., 2011).

As the pre-colonization interval does not show physical evidence of the interaction between the insects and the corpse, it is not possible to estimate the pre-CI accurately. Also, the pre-CI is often not discussed in literature. When a body is instantly exposed to the environment, the precolonization phase is generally short, but in many forensic cases bodies are artificially hidden and preserved from insect activity, delaying the subsequent insect colonization (Tomberlin et al., 2011). Bodies can be buried, wrapped in clothes or blankets, burned or hidden inside buildings and cars. Also, habits and characteristics of the dead person itself, such as alcohol or drug abuse, diet and genetics can have an influence on the volatiles that are produced after death. It is therefore very important to get more insight into factors affecting the time between the death of a person and the colonization of insects. If the precolonization period is ignored or estimated incorrectly, this can have large consequences for the criminal investigation.

1.2 Phases in the precolonization interval

In the pre-colonization interval three phases are recognized: [1] the exposure phase, [2] the detection phase and [3] the acceptance phase (Figure 1) (Tomberlin et al., 2011). The exposure phase represents the time between the death of the animal and the exposure of the carcass to insect detection. As already mentioned, this period can be very short, but also longer, when remains are hidden and preserved. For example, when a body is stored in a cold chamber of a morgue, it can be said that the corpse is not yet exposed to insect detection. The detection phase, in which the insects receive chemosensory input from the carrion, is further divided into activation, the detection of decomposition cues by insects for the first time, and searching, the time the insects need to find the carrion after sensory activation. The searching behaviour of arthropods is regulated by the idiothetic control system to process internal stimuli and the allothetic control system, which processes external stimuli. Thus, the response of insects to food sources is determined by external factors, such as temperature and time of day, and internal factors such as ovarian development (Tomberlin et al., 2011). Together, internal and external factors determine the behaviour of the insect after chemical substance detection. For example, Brodie et al. (2016) showed a different attraction of green bottle flies (Lucilia sericata) to fresh faeces (feeding site) and carrion (oviposition site) for gravid and non-gravid females. Where non-gravid female green bottle flies, not fed

Figure 1 | Proposed entomological association with carrion. Modified from Tomberlin et al. (2011) and Rivers & Dahlem (2014)

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6 for three days, were evenly attracted to faeces and carrion, well-fed gravid females were only attracted to carrion.

The third phase in the precolonization interval is the acceptance phase, the time between the first physical insect-carrion contact and the establishment of residence of the insect on the carrion. Insects use cues such as shape, colour, size and taste of the source to identify it and determine its suitability as a food source or oviposition site. Also, chemicals produced by other organisms, such as predator insects (feeding on the necrophagous species) or competing insects, influence the behaviour of insects during the acceptance phase (Tomberlin et al., 2011). The postcolonization interval is divided into the consumption phase, the time between the initial colonization of the insects and departure from the carcass, and the dispersal phase, the movement and departure of the insects. As this thesis focuses on the initial detection of carcasses by insects, the postcolonization interval will not be further discussed (Tomberlin et al., 2011).

Chapter 2: How do flies detect and process chemicals?

Just like humans, insects use different sensory input (smell, taste, touch, sound and sight) to obtain information about their environment. Compared to humans, insects are much more sensitive to chemicals in their environment (Rivers & Dahlem, 2014). Insects mainly rely on chemical stimuli for the finding of mates, food and oviposition sites (Rivers & Dahlem, 2014; Weiss & Atwood, 2011). Apneumones, chemical substances produced by non-living objects such as decomposing animals, and pheromones, used for the communication between insects, are examples of these chemical stimuli (Rivers & Dahlem, 2014; Weiss & Atwood, 2011). However, these chemical substances do appear as complex mixtures in the air. Insects need to detect and identify many different chemical substances associated with mates, food and oviposition sites and predators, but also the concentrations of these substances can be important, as they can give information of the nutritive value and the proximity of food sources (R. M. Joseph & Carlson, 2015). According to Rivers & Dahlem (2014) chemical compounds form concentration gradients in the air, with the highest concentrations present close to the source and decreasing concentrations as you get further away, which are used by insects to locate the source they are looking for. However, according to Murlis & Jones (1981) odour plumes do not exist in continuous gradients, but appear in odour bursts intermitted with spaces not containing any odour. Therefore, insects have to average chemical stimuli they detect to follow the general increasing concentrations when they get closer to the source. Necrophagous insects will first detect long-distance decomposition cues such as volatile compounds in the air and body fluids. Shorter-distance cues, such as low-volatility compounds, are used to detect the carrion when they come closer to the source (Tomberlin et al., 2011). Different insects are sensitive for different compounds, which explains the succession waves of different arthropods on carrion over time, as the proceeding decomposition process results in the production of different volatiles over time (Tomberlin et al., 2011).

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7 Flies can detect carrion from several kilometres away, showing that this attraction is highly dependent on chemical cues rather than visual cues (Frederickx et al., 2012). For the detection of chemical compounds insects use two forms of chemoreception, taste (gustation) and smell (olfaction). Therefore, in this thesis I will focus on chemoreception of insects rather than the other sensory input such as vision and sound.

2.1 Chemoreception

For insects it is highly important to detect and respond on the chemical world surrounding them, for example for the finding of food and mates. Insects have two ways to detect chemicals in their environment: olfaction and gustation. Olfactory receptor neurons (ORNs) are responsible for the detection of volatile substances, whereas gustatory receptor neurons (GRNs) are used to detect non-volatile substances (R. M. Joseph & Carlson, 2015). Insect olfaction is used to detect interesting sources; gustation to test sources when they are found and to decide whether the sources are accepted or rejected (Scott, 2018). The location of the chemical receptors on the body of the insect is essential for the sensitivity of chemical detection. The chemoreceptors associated with olfaction are commonly located in anterior regions such as the head, antennae and prothorax (anterior part of the thorax), in a forward position relative to locomotion (Rivers & Dahlem, 2014). The chemoreceptors associated with gustation are located on the labellum (feeding organ of flies), but also on the wings and legs, allowing the insect to taste potential food or oviposition sites before using the mouthparts (R. M. Joseph & Carlson, 2015). According to Rivers & Dahlem (2014) insect olfaction requires many different types of receptors, but insect gustation only detects broad groups of compounds, distinguishing only between sweet and bitter. However, Joseph & Carlson (2015) mention that insect gustatory receptors are also sensitive to salt levels and water. Scott (2018) shows that many different genes associated with gustatory receptors exist, suggesting that the sense of taste of insects is more complex that currently assumed.

Chemical receptors, also called sensilla, are generally fine thin hairs (setae) containing one or more pore openings for chemical molecules to enter (Figure 2). However, some sensilla show a different morphology, such as a plate- or peg-like morphology. Different types of chemoreception are associated with different types of sensilla, with a certain morphology and number of pores. Sensilla associated with olfaction usually have a hair-like morphology and contain multiple pore openings along the walls; gustatory receptors appear in different morphologies and contain only one pore for chemical penetration. The sensilla are associated with sensory neurons, which dendrites are lying in close

Figure 2 | Basic structure of an olfactory sensillum. Modified from Weiss & Atwood (2011)

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8 proximity to the walls and pores of the receptor (Rivers & Dahlem, 2014). When volatile substances enter the pores in the sensilla, they enter the sensillum lymph, the fluid surrounding the dendrites of the olfactory sensory neurons (OSNs). The volatile compounds bind odorant binding proteins (OBPs) in the sensillum lymph, which transport these molecules over the sensillum lymph to the nerve receptor sites at the dendrites of the sensory neurons. In one sensillum, dendrites of approximately one to four OSNs are present (Weiss & Atwood, 2011). For further action to happen, a certain threshold must be surpassed, often induced by the binding of several molecules to several similar receptor proteins at the same time (Rivers & Dahlem, 2014; Weiss & Atwood, 2011). The signal will be transferred to the neuronal cell body, causing further actions in the brain to happen (Rivers & Dahlem, 2014; Weiss & Atwood, 2011).

Summarizing, insects live in a chemical world rather than a visual world (such as humans) and are dependent on volatile compounds in the air for all important activities in their lives: the finding of food, mates and oviposition sites. Both gustation and olfaction play a role in this. The olfactory and gustatory chemoreceptors are located in such a way that these chemicals can be detected and tasted. Insects of the same species can react differently on chemical substances, as also internal factors (such as hunger and pregnancy) play a role in the behaviour following substance detection.

Chapter 3: What volatile compounds are produced by cadavers?

During decomposition multiple volatile compounds are produced. Before going into more detail about the chemical substances that are released by decomposing carcasses, it is important to have some background knowledge of the process of human decomposition in general.

3.1 Human decomposition

Decomposition takes place from the moment of death until the moment that the body is reduced to a skeleton. Although this is a continuous process, this process is often divided into five consecutive stages (Goff, 2009). During the [1] fresh stage, beginning at the time of death, some early changes to the body take place: the body temperature will decrease (algor mortis), muscles stiffen (rigor mortis) and the abdomen will get a greenish discoloration (Goff, 2009; A. Vass, 2001). Female flies (Calliphoridae and Sarcophagidae) will arrive at the body, explore the body and deposit eggs in body openings, such as eyes, genital areas and wounds (Goff, 2009). When the bacteria inside the body start to digest the tissues, gases are produced, which cause the abdomen to inflate. This is called the [2] bloated stage. The bloated stage is followed by the [3] decay stage, when the body is further broken down by insects and bacteria resulting in gases escaping the body, and the [4] post-decay stage, when the body is reduced to skin and bone. During the [5] skeletal stage, only bones and hair are left (Goff, 2009).

Decomposition starts with the self-digestion of the cells of the body, by enzymes released from the cells themselves. Following the shortage of oxygen in the cells, after the heart stops beating, body cells rupture and release their cellular enzymes, such as proteases and lipases. These enzymes start to attack and degrade the cells in the body. This process is called autolysis and begins within minutes after

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9 death (Rivers & Dahlem, 2014; A. Vass, 2001). Autolysis is followed by putrefaction, the degeneration of soft tissues of the body by microorganisms, mainly bacteria. This process starts in the abdomen, as most bacteria are present in the intestines. It is during this process that all kinds of molecules, liquids and gases are formed by microorganisms and released (Rivers & Dahlem, 2014; A. Vass, 2001). All body tissues, containing proteins, fats and carbohydrates, will be broken down, resulting in the production of different compounds and release of gases in the air (A. Vass, 2001).

3.1.1 Protein decomposition

The breakdown of proteins, which starts during autolysis, is called proteolysis and is done by cellular enzymes and microorganisms. Not all protein-containing body tissues degrade at a similar rate; epithelial and neuronal tissues degrade relatively fast, epidermal and collagen tissues relatively slow. Keratin, which is present in high amounts in skin and hair, contains a high number of disulphide bonds between cystine structures, making it highly resistant to proteolysis. This explains why hair remains for a long time during decomposition, often still present on skeletonized carcasses (Dent et al., 2004; Rivers & Dahlem, 2014). The rate in which proteins are degraded, is dependent on the availability of oxygen, the moisture content, the protein composition and the enzyme content and abundance within the tissue (Rivers & Dahlem, 2014). During the breakdown of proteins different chemical substances are formed, such as phenolic compounds skatole and indole, and gases hydrogen sulphide, methane, ammonia and carbon dioxide (Dent et al., 2004; Rivers & Dahlem, 2014; A. Vass, 2001). Amino acids containing sulphur, such as cysteine and methionine, are further processed by bacteria and produce sulphur-containing gases hydrogen sulphide gas, ammonia, thiols, sulphides and pyruvic acid (Dent et al., 2004).

3.1.2 Fat decomposition

The degradation of body fat involves hydrolysis by cellular lipases and bacterial enzymes. The hydrolysis of lipids results in formation of saturated and unsaturated fatty acids, and low amounts of hydroxy-fatty acids. When hydrolysis proceeds unsaturated fatty acids are converted to saturated fatty acids, resulting in high amounts of saturated fatty acids and low levels of unsaturated fatty acids when decomposition proceeds. Eventually, when enough water is present and lipid hydrolysis proceeds, all the original body fat will be reduced to a mass of saturated fatty acids (Dent et al., 2004; Rivers & Dahlem, 2014). Under certain circumstances, these fatty acids can be converted into adipocere, a process called saponification. Adipocere is a product formed by fatty acids, forming a yellowish-white wax-like substance surrounding the corpse. Saponification generally takes place under warm and moist conditions (Rivers & Dahlem, 2014; A. Vass, 2001).

3.1.3 Carbohydrate decomposition

The breakdown of carbohydrates starts with the bacterial breakdown of glycogen into glucose molecules in the liver. Dependent on the oxygen availability the glucose monomers are further processed by microorganisms. During the beginning of decomposition, when plenty of oxygen is present, most of the glucose molecules will be oxidized to form carbon dioxide and water. Some glucose molecules are used by bacteria which convert the glucose molecules into organic acids and alcohols. When oxygen levels drop as decomposition proceeds and anaerobic conditions arise and oxidation is no longer possible, the

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10 glucose molecules are converted into lactic acid, butyric acid, alcohols, hydrogen sulphide and methane. When soft tissues are further degraded and more air movement in body cavities takes place, more oxygen is available again. Aerobic pathways will take place again, creating more carbon dioxide and water (Dent et al., 2004; Rivers & Dahlem, 2014).

3.2 Volatile compounds released during decomposition

During decomposition many different compounds, liquids and gases are produced (Rivers & Dahlem, 2014). Paczkowski & Schütz (2011) compared different studies into volatile decomposition compounds. They concluded that the formation of volatiles during decomposition is very complex, depending on many different factors and showing a lot of variation. In this chapter some studies done into volatiles produced during decomposition will be discussed and compared. Studies into human remains, as well as some studies into animal carcasses, will be discussed.

3.2.1 Methodology

Research into human decomposition is needed to improve criminal investigations and medical examinations of human corpses. However, few bodies are available for forensic research into this topic, causing very low numbers of subjects (n=1 or n=2) to be used. Because the availability of bodies is unpredictable and the human cadavers are often not comparable (such as having different body masses), the results of these studies are not statistically valuable. So, these case studies are interesting, but not useful to draw hard conclusions. To circumvent this problem, animals are used as surrogate models for human bodies, making it possible to use a bit more (comparable) subjects per research and increase sample size (Matuszewski et al., 2019). Especially pigs are used extensively. However, due to ethical reasons, still not many animals can be used. More important, it is unclear how well these surrogate models can be used to be applied to human decomposition. Another problem is a lack of facilities for decomposition research, and that these facilities are spread over the world. Consequently the results of repeated experiments are compromised by environmental differences caused by climate, seasonality and habitat.

When focussing on research into volatiles forming during decomposition, the results are dependent on the abiotic factors affecting the release of volatiles from the carcass, but also the sampling technique and subsequent analytical procedure. Volatile samples can be taken passively or actively, for example when using an active gas flow. Solid phase microextraction (SPME) is a technique used for sampling of volatiles in a static headspace. A fibre with (specific or non-specific) adsorption material is exposed in the sample headspace and inserted into a gas-chromatography system for analysis. The longer the exposure time of the fibre in the headspace, the more volatile compounds are adsorbed. For sample substances that are not formed in high levels, an active air stream is needed (Paczkowski & Schütz, 2011). Many studies into decomposition volatiles use a combination of gas-chromatography (GC) and mass spectrometry (MS) to analyse the air samples. In the gas chromatograph the sampled compounds are being separated based on their affinity for the substance (stationary phase) in the column. Thereafter the molecules enter the mass spectrometer, which detect the separate molecules by their mass-to-charge ratio. This allows the researchers to determine which VOCs are present.

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3.2.2 Human

Hoffman et al. (2009) investigated volatile organic compounds (VOCs) that were released during decomposition of 14 pieces of human tissue: blood, muscle, testicle, skin, bone, adipocere, fat (adipose) tissue, bone and teeth. The collected tissue samples were placed in vials and the headspace was then analysed using solid-phase microextraction (SPME) to extract the volatiles present, and gas chromatography-mass spectrometry (GC-MS) for substance identification. In total, 33 VOCs were detected, which were divided into seven chemical groups: acids/acid esters, alcohols, aldehydes, halogens, aromatic hydrocarbons, ketones and sulphides. Some of the detected VOCs were dimethyl disulphide, toluene, heptanal, octanal and nonanal. The tissues that showed the highest number of VOCs were fat tissue (22 VOCs), muscle (19 VOCs), bone (19 VOCs) and adipocere (18 VOCs), compared to testicle (9 VOCs), teeth (6 VOCs) and skin (5 VOCs). However, no statistical analysis was done. No compound was produced by all tissue types. However, p-Xylene was present in 13 of the in total 14 samples (only absent in teeth tissue) and tetrachloroethylene and 2-pentul-furan were present in 9 of the 14 samples. Some compounds were produced by only a few tissues, such as propanoic acid (2/14), hexanoic acid (2/14), 2-Hexenal (3/14) and nonanal (3/14). This research suggests that all human tissues show a different chemical profile, with some compounds being shared by other tissues and some compounds being relatively unique.

In another study by Statheropoulos et al. (2005) the chemical profile produced by two dead men found in the Mediterranean sea three to four weeks after death was investigated. Putrefaction was taking place in both cadavers. To measure VOCs a hole was made in the body bags at the upper chest levels of the bodies inside. Then, 5L of air was pumped through a sampling tube using a sampling pump. For each body bag, also a reference sample, outside the body bags, was taken. The air samples were analysed by a combination of thermal desorption, to concentrate the VOCs before entering the gas chromatograph, and GC-MS. In total more than 80 compounds were identified. The compounds that were present in the highest concentrations were dimethyl disulphide (13.39 nmol/L), toluene (10.11 nmol/L), hexane (5.58 nmol/L), benzene 1,2,4-trimethyl (4.04 nmol/L), 2-propanone (3.84 nmol/L) and 3-pentanone (3.59 nmol/L). On both bodies DMDS was found to be the most abundant. In lower concentrations also other sulphur-containing compounds, such as methyl ethyl disulphide (0.96 nmol/L), carbon disulphide (0.85 nmol/L) and dimethyl trisulphide (0.67 nmol/L), were found. As already mentioned, these sulphur-containing compounds are produced during degradation of proteins. Also a high amount of esters was found on the cadavers, probably as a result of saponification. Although this research gave more insight into the formation of volatile compounds during decomposition, the bodies were found in the water and were already dead for three to four weeks, which makes the research quite specific and limited. Also, measurements were done at only one time point in decomposition.

Statheropoulos et al. (2007) focused on the formation of VOCs in the more early stages of decomposition. Four days after death the produced VOCs of a human body were monitored for 24 hours. The body was placed in a sealed body bag, a sample tube was inserted and 5L of air was sampled with the use of a pump. Samples were taken at 0h, 4h, 8h and 24h. In total 30 substances were identified. Of these 30 substances, eleven of them were present at all four sample moments and are considered core

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12 substances of decomposition, among which 2-propanone and dimethyl disulphide (DMDS), also found in the research of 2005. Also, ethanol, methyl benzene, octane, dimethyl trisulphide and xylenes were present throughout the sampled 24h. Statheropoulos et al. (2007) also found that DMDS was the most prominent sulphur-containing substance. Different classes of substances were formed at different rates and at different moments in the decomposition process. For example, p-Xylene levels increased from 0.003 to 0.058 (substance gradients) between 0 and 24 hours, whereas ethanol levels increased from 0.010 to 0.375 between 0 and 4 hours and decreased to 0.002 between 4 and 24 hours. These different production rates possibly correlate to the rate at which different tissues are getting degraded by microorganisms.

The final research into volatile compounds produced by human cadavers I want to discuss, is the research of Vass et al. (2008). During this study four bodies were buried in shallow graves (0.46-1.07 m depth) and the volatiles appearing at the surface were monitored for four years. During this research no less than 478 volatile and semi-volatile compounds were identified by GC-MS. From these 478 compounds, 30 were selected as being key markers of human decomposition, such as carbon tetrachloride, nonanal, naphthalene and benzene. Interestingly, these key markers of human decomposition show overlap with previous mentioned research, which also identified toluene, hexane, carbon disulphide and dimethyl disulphide. However, this research looked into the compounds produced by buried remains rather than bodies found on the surface. Therefore, it is important to be aware of the fact that the formation and distribution of volatiles from buried remains can be different from that of remains laying on the ground. In chapter 4 the effect of burial on compound formation and distribution will be further discussed.

Although these four studies identified the volatiles formed during decomposition of human remains, they used different tissues and bodies in different conditions and different methods. This makes it hard to compare them. The discussed studies show that the process of VOC formation during decomposition is very complex, giving rise to many different compounds. However, some compounds were repeatedly found in multiple studies. Substances found multiple studies were toluene, nonanal, dimethyl disulphide, dimethyl trisulphide, hexane and carbon disulphide. Sulphide-containing compounds, especially DMDS, appear to be the most consistent throughout all studies. Further research must be done into the formation of volatile compounds during decomposition to investigate this further. However, based on current knowledge sulphide-containing compounds seem to be produced during decomposition relatively independent of study conditions and methods.

It is worth mentioning that these studies order the detected volatile compounds purely on quantity, after which the most abundant substances are characterized as key markers. It is important to realize that this classification therefore does not provide any information about the biological relevance of the compounds.

3.2.3 Animals

Dekeirsschieter et al. (2009) studied the VOCs formed during the decomposition of six pig carcasses. In three different biotopes (a forest, an agricultural and an urban site) two pig carcasses were placed and

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13 monitored. During this research in total 104 VOCs were identified, which could be divided into the chemical classes acids, esters, ketones, aldehydes, alcohol, nitrogen compounds, sulphur compounds, halogens, cyclic and non-cyclic hydrocarbons. All three biotopes showed a different chemical profile, with some volatiles being specific for one biotope, but also many volatiles being present in all three biotopes. Also these researchers selected a group of (35) key compounds, again containing dimethyl disulphide. In this study also the development of volatiles during different decomposition stages was investigated. During the fresh stage, no VOCs were detected. During the bloated stage different sulphur-containing compounds, such as DMDS, dimethyl trisulphide and sulphur dioxide, and alcohols were formed. During the decay stage still sulphide-containing substances and some cyclic compounds (indole, phenol and 4-methylphenol) were produced. During advanced decay production of aldehydes was measured.

In 2016 more research was done into VOCs produced by pig carcasses (Armstrong et al., 2016). In this study three pig carcasses were placed outside and monitored the following 72 hours. In total 105 volatile compounds were identified. From these 105 compounds, nitrogen- and sulphur-containing compounds (dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide) showed the highest proportion of detected VOCs (25 and 24 VOCs respectively) and were present in all samples. Besides the sulphur-containing compounds, also nitrogen-containing compounds can be produced during protein degradation. When looking into the formation of compounds during the time after death, esters were the most abundant directly after death; esters and ketones 17-23 hours after death; sulphur containing compounds 43-49 hours after death and sulphur-containing compounds and carboxylic acids 69-75 hours after death.

In 2012 Paczkowski et al. investigated the volatiles produced by five mice carcasses. In this research 11 compounds were identified, including some compounds also found in earlier mentioned studies: dimethyl disulphide, nonanal, phenol and indole. Nonanal was mainly found during the fresh stage of decay (1-52 ng/µL) and in low concentrations during later stages (1-12 ng/µL). Sulphur-containing compounds dimethyl disulphide and dimethyl trisulphide were mainly found during the active stage decay (in concentrations of 1-60 ng/µL and 1-104 ng/µL respectively). When comparing the decomposition of dead mice to human carcasses, the decomposition of small cadavers appears to be much faster than decomposition of larger cadavers. This is an important factor needed to be taken into account when using research into decomposition of small animals, such as mice, for the prediction and study of human decomposition.

In studies into human decomposition often carcasses of pigs are used, as generally not many human bodies are available or cannot be used because of ethical reasons. Pigs are believed to be the most similar to humans, having similar internal organs, gut fauna, muscular structure, hairless skin and chemical composition (Statheropoulos et al., 2011). However, Cablk et al. (2012) showed that the volatile profile produced by pig carcasses is the most different from humans, when comparing it to cow and chicken carcasses. In this study, volatile samples produced by four different tissues (bone, muscle, fat and skin) of cow, pig and chicken carcasses were compared to the compounds found in the study of Hoffman et al. (2009) into volatiles produced by human remains. Nonanal was found in all decomposed

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14 samples, present in bone, fat and muscle, but absent from fresh skin. When comparing the VOCs produced by cow, chicken and pig tissues with human, chicken and human appeared to be the most similar (in the total number of produced compounds and the number of compounds present in chicken as well as human). Human and pig appeared to be the least similar. Nonanal, toluene and xylene, some compounds already mentioned earlier, were produced by all four organisms. Also in this study dimethyl disulphide was found, but only in chicken and cow, not in the pig carcasses. As this compound appears to be present in high amounts during human decomposition, it is remarkable that exactly this substance is not found during decomposition of pig carcasses in this study. When looking into the different tissue types contributing to the VOCs produced, the three animals show more similarity with each other than with human (Figure 3). This comparative study confirms that awareness is needed when model organisms are used to study VOC formation during human decomposition.

In this chapter, different studies are discussed focusing on the formation of volatiles during human and animal decomposition. The comparison of volatiles produced by decaying animals and humans show that there are many similarities. Substances that are found in human as well as animal studies are: dimethyl disulphide, dimethyl trisulphide, nonanal and carbon disulphide. It is clear that sulphide-containing compounds, such as DMDS, DMTS and carbon disulphide, are produced during active decay, but not directly after death. However, it is remarkable that several compounds are measured in some studies, and not detected in others. Also the amount of volatiles found was highly varied between studies. This could be caused by the use of different techniques or analyse procedures. However, all studies use GC-MS to identify the chemical compounds. It is also possible that internal differences between the subjects cause some differences in produced volatiles, whereas the decomposition of general tissues such as muscle and fat tissue lead to the common found volatiles. These differences make it hard to pinpoint which exact compounds are produced by which tissues and when.

Figure 3 | Relative contribution of bone, fat, muscle and skin tissue to the VOCs formed. Modified from Cablk et al. (2012)

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Chapter 4: What volatile compounds are detected by flies?

Now we know what types of volatiles are produced by cadavers, it is important to know which ones can be detected by Diptera. Therefore, the responses of flies on different compounds need to be studied. When investigating the olfactory response of insects to volatile compounds, often wind tunnel experiments are performed. A wind tunnel is a tube or box with air flow which can be used to study the flight or walk behaviour of insects, when odours are added (Knudsen et al., 2018). Also, electroantennographic experiments can be done, in which the olfactory stimulation of insect antennae is recorded. An electroantennogram (EAG) measures the change of potential between the antenna tip where the odours enter and the base of the antenna in response to a volatile substance (Chakravarthy & Selveanarayanan, 2019). This technique measures the response of the olfactory receptors on the chemical substances added.

Paczkowski et al. (2012) investigated which compounds induced a response in the antennae of female blow flies Calliphora Vicina by electroantennographic experiments. In total seven compounds were identified which were electrophysiologically active: dimethyl sulphide (DMDS), dimethyl trisulphide (DMTS), nonanal, hexan-1-ol, 1-octen-3-ol, 3-methylbutan-1-ol and heptanal. Therefore the test substances were diluted in paraffin oil in concentrations ranging from 10-9_{to 10}-2_{mg/mg. Filter paper} was drenched with these dilutions, placed in glass syringes and 2.5 mL of the air in each syringe was passed over the female C. Vicina antennae. DMDS and DMTS were detected by the antennae at very low concentrations (of 10-9_{and 10}-8_{respectively). Although the direct behavioural effect of these} sulphur-containing compounds is not tested, these results suggest that both substances are important cues for the blow fly to detect carrion. However, it is known that C. Vicina blow flies are present on cadavers within minutes after death. As sulphur compounds are not produced directly after death, they cannot play a role in initial detection of cadavers. The only compound produced directly after death was nonanal, a substance also mentioned by earlier discussed literature (Cablk et al., 2012; A. A. Vass et al., 2008). Nonanal is produced during the early degradation of fat on skin and fur. This suggests that possibly this substance is used by flies to initially detect dead vertebrates in the area. However, no wind tunnel or EAG experiments are carried out yet to study this substance further.

Johansen et al. (2014) investigated the response of C. Vicina on mouse carcasses. In this study wind tunnel experiments were performed to investigate the oriented flight of male and female blow flies during the process of decay of dead mice. Also the volatiles produced by the mouse carcasses were analysed using GC-MS. The percentage of oriented flight increased from 36% using fresh cadavers up to 68% using three days old cadavers. The percentage of oriented flights stayed relatively constant after this, with 61% using six days old cadavers and 65% using nine days old cadavers. Between 9 and 33 days the cadavers showed to be less attractive, with oriented flights around 45%. During this study in total 23 compounds were identified, of which nine showed the highest amounts during the most attractive stages of decay. Butylated hydroxyl toluene, 3-hydroxy-2-butanone, 2-phenyl ethanol, 3-methyl butanol, dimethyl trisulphide, nonanal, phenylacetaldehyde, 3-octanone and dimethyl disulphide all showed an increase in concentration from day 1 towards day 9 and a decrease in concentration later on. From these

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16 nine compounds, DMDS showed the highest levels, of 315 and 294 (relative to the internal standard heptyl acetate) on day 6 and 9 respectively. In contrast to the research of Paczkowski et al. (2012), nonanal was not yet present during the fresh stage. This research suggests that, although some blow flies appear early in decomposition, the highest response is found for carcasses of three to nine days old, corresponding to high amounts of dimethyl disulphide and dimethyl trisulphide.

A study using male and female Lucilia sericata (Calliphoridae) looked into the behaviour of flies in an Y-tube (two-arm) olfactometer in response to seven VOCs produced by decomposing carcasses (Frederickx et al., 2012). Two doses of each substance, 100 µg and 0.05 µg, were tested. The substances were applied on small pieces of filter paper and placed at the end of one of the two tubes, with a control filter paper at the end of the other arm. Continuous airflow with a rate flow of 800 mL/min was maintained through the olfactometer with use of a pump. For each chemical compound fifty female and fifty male flies were placed at the main branch of the olfactometer, giving them the opportunity to enter one of the two arms. The highest responses were induced by low concentrations (0.05 µg) of DMDS and butan-1-ol. However, very high concentrations (100 µg) of these substances did not induce a reaction. This can be explained by the fact that such high levels of these substances are naturally not produced by cadavers and therefore flies do not associate these high concentrations with interesting sources. Also no response to phenol and indole was seen. An interesting result was that female flies were more sensitive to the VOCS than the male flies.

It is clear that sulphide-compounds, such as dimethyl disulphide and dimethyl trisulphide, which are produced during protein degradation, are important cues for blow and flesh flies for the detection of cadavers. The formation of nonanal is less clear, as not all cadavers produced this for unknown reasons. Paczkowski et al. (2012) found nonanal directly after death, suggesting a role of this compound in initial cadaver detection by insects, whereas Johansen et al. (2014) did not detect nonanal during this stage. Johansen et al. also showed that, although 36% oriented flight was measured during early decomposition, most insect attraction took place between three and nine days after death, with an oriented flight of around 65%. It is possible that, as compounds detected by insect antennae only appear after a couple of days, the initial attraction of insects in natural circumstances is also driven by visual cues or by chemical compounds secreted by insects already present on the carcass. However, this does not explain why still 36% of the flies were attracted towards the mouse carcasses, as this was a wind tunnel study, making the response on visual cues impossible. However, the methodologies of this study are not entirely clear. It is unclear how these percentages were measured. If every flight directed towards the volatiles is mentioned as an ‘oriented flight’ also random flight behaviour of these insects can be detected as oriented flights.

Sulphur-containing compounds, produced by cadavers during active decay, are detected by fly antennaein very low concentrations (10-9_{and 10}-8_{mg/mg paraffin oil), according to EAG experiments} (Paczkowski et al., 2012). The question is whether these compounds play a reasonable part in the first detection of cadavers by flies. It makes sense that these compounds are important for insects to be detected, as it is a sign of the beginning of active decay, during which still large amounts of soft tissue are present for larvae to develop upon and reach adulthood. This makes the corpse a suitable oviposition

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17 site. Also, the presence of high amounts of volatile compounds implicates that body openings are present (from which these compounds escape), which are suitable oviposition sites. Although high levels of sulphide-compounds are only produced a few days after death, it is possible that very low amounts of these compounds are already produced earlier in decomposition. The extreme sensitivity of flies for these compounds could allow flies to detect carrion far before active decay begins and arrive at the corpse before high amounts are formed. This would make sense, as in this way insects arrive at carrion when a maximal level of nutrients is present.

The study of Frederickx et al. (2012) also showed a behavioural response of flies towards low concentrations of DMDS, and another substance: butan-1-ol. However, very high concentrations of these substances (100 µg) did not induce a response. This can be explained by the fact that these high levels are only produced when the cadaver is in an advanced state of decomposition and not much soft tissue is remaining, making the cadaver a less suitable oviposition and food site.

In these studies, often electroantennographic and wind tunnel experiments were conducted, to measure the (behavioural) effect of volatiles on flies. However, although some specific volatile compounds showed a response in fly antennae and these compounds were also present in volatile mixtures inducing fly attraction in wind tunnel experiments, the direct behavioural attraction of flies towards these volatile compounds were not tested. Also, compounds present in the highest concentrations were assumed to play a role in the detection of cadavers by Diptera. However, as shown in chapter 2, flies possess very sensitive olfactory receptors, which can detect very low concentrations of substances. It is therefore certain that also substances in low concentrations can be detected by flies. So, also substances which appear in much lower concentrations can play an important role in fly attraction.

Lastly, the fact that a chemical compound is electrophysiologically active does not automatically mean that this substance induces a behavioural response in flies. It is possible that this substance only induces behavioural changes if the mixture and concentrations are right.

Chapter 5: What factors affect attraction of Diptera towards carcasses?

The model used to understand and predict insect activity on corpses is based on terrestrial cadaver decomposition during seasons with high temperatures and therefore high insect activity. The insect succession on vertebrate cadavers in this environment is relatively predictable and used to do PMI estimations. However, in daily life the locations where people die or where bodies are placed do not resemble these ‘ideal’ circumstances. The insects that are associated with these bodies and the subsequent insect succession will therefore differ in many aspects from this terrestrial carrion decay. Some of the processes that can be altered by circumstances are the detection of the cadaver, the oviposition and development of the insects (Rivers & Dahlem, 2014). The artificial conditions that can be associated with criminal cases, such as body alteration (burning, dismemberment, packing), can have a large impact on the initial detection of the body by insects and eventually lead to wrong PMI estimations. Research is done into the effect of various abiotic factors, such as temperature, humidity and pH on the rate and

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18 process of decomposition. These factors will not be discussed in this thesis. The focus of this chapter will be on different factors specifically inhibiting or accelerating the cadaver detection by Diptera.

5.1 Physical barriers

Physical barriers can hold back the spreading of chemical substances, making them indetectable for flies. Different physical barriers can exist. Bodies can be located indoors, stuffed in bags, wrapped in blankets or hidden in containers or vehicles (Campobasso et al., 2001; Rivers & Dahlem, 2014). The volatile compounds emitting from these cadavers will only escape through small openings, which extends the diffusion time and so is the time insects need to find it. In some situations, such as when bodies are tightly packed into plastic, no gases will escape until the accumulated gases force the material to rip or break and volatile compounds are released in high concentrations at once. The spread of VOCs in the environment depends on thermal energy and is therefore dependent of the ambient temperature. The exact delay of insect detection and attraction is dependent on the specific conditions of each case, making it difficult to predict which effects certain barriers will have (Rivers & Dahlem, 2014).

A natural example of a physical barrier is when bodies are buried. Shallow burials may only delay the insect detection for a short time, whereas deep burials may abolish volatile compound release at all. This is also dependent on the soil type and water content of the soil. Soil that is loosely packed, such as sand, permits more air movement than soil consisting of tightly packed particles, such as clay (Rivers & Dahlem, 2014). Rodriguez and Bass (1985) studied the decomposition and insect activity of buried human cadavers. During this study six bodies were buried at different depths (0.3m, 0.6m and 1.2m) within 48 hours after death. During the experiment thermometers were placed above ground and within the graves. Also, insect species present in and on the graves were identified. This study showed that the decomposition of the buried corpses went slower compared to corpses placed above ground (discussed in earlier studies of the same researchers), caused by lower temperatures and limited insect access. The bodies placed at 0.3m showed some insect activity, caused by the oviposition of eggs from adult blow flies in soil cracks above the grave, and subsequent movement of the larvae towards the corpses. Apparently, the volatile compounds produced by the cadavers were detectable by different species of carrion insects when they were buried at 0.3m depth. Corpses placed deeper in the ground were not discovered by insects. All insects present in and around the graves were identified as Calliphoridae (blow flies) and Sarcophagidae (flesh flies). Beside the inhibited insect activity on corpses and the lower temperatures, also the decreased availability of oxygen in burials slows down the decomposition process of buried remains (A. A. Vass et al., 2008).

Another example of a physical barrier is when bodies are in an aquatic environment. In general bodies will sink first, until so much gas is formed inside that bodies start to float. At this point the bodies are generally already discovered by marine insects and animals and less soft tissue will be present. It is just from the moment that bodies are floating, and are located close to land, that they can be detected by terrestrial flies (Campobasso et al., 2001; Rivers & Dahlem, 2014).

Even when a cadaver is detected by insects, openings permitting gas escape in the environment may be too small for insects to enter. In these cases, often numerous dead flies are found at the points

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19 were volatiles escape, due to their attempts to reach the cadaver. Even sometimes eggs or larvae are laid in these openings (Rivers & Dahlem, 2014).

Also clothing on a body can be considered a physical barrier. Mann et al. (1990) describes that clothing on a corpse can enhance the decomposition process. By slowing down the cooling of a body, keeping the corpse warmer and more humid the corpse is more attractive to flies and thereby the rate of decomposition is increased. On the contrary, Mashaly et al. (2019) found no significant effect of clothing on the decomposition rate and presence of insects on rabbit cadavers. For this study 18 mature rabbit carcasses were used, from which nine rabbits were covered with clothes resembling human clothing (a cotton shirt, shorts and underwear) and nine were left unclothed. The rabbits were placed outside on the ground and the attracted insects were trapped for six days using a plastic tray containing water, soap and salts. Then, the collected insects were identified. This research showed no significant difference in the rate of decomposition and the presence of insects between the clothed and unclothed rabbit carcasses.

5.2 Burning

Bodies, associated with forensic investigations, can be severely burned or charred, in order to prevent identification of the body and the discovery of physical evidence. Campobasso et al. (2001) mention that burned or charred human remains are less attractive for Diptera, because these remains contain less protein rich tissues and less water, both needed for larvae to grow and develop. However, Rivers and Dahlem (2014) suggest that the attraction of flies towards burned human cadavers is dependent on the severity of the damage of the tissues. The severity of physical injuries by burning can be classified by the Crow-Glassman Scale (CGS) which contains five levels of tissue damage (CGS1-CGS5) with increasing levels of damage. Bodies showing injuries consistent with CGS1 and GCS2, such as damage caused by severe smoke inhalation and some destruction of the feet and hands, appear to be more attractive to insects than unburned human remains. This is caused by the leakage of fluids and gases from the abdominal area and wounds to the skin. Bodies with severe tissue damage, consistent with GCS3-GCS5, appear less attractive by flies, because the low moisture contents are not favourable for larval development. Also research of Introna et al. (1998) into the insect succession on charred bodies (in cars) showed early Diptera colonization as flies were attracted towards the smell produced by the exposed inner cavities.

5.3 Chemical modifications

Besides the physical barriers that can impair insect attraction, also chemicals used on bodies may affect the attractiveness of corpse to insects (Rivers & Dahlem, 2014). Charabidze et al. (2009) studied the effects of multiple household products on the attraction of flies towards rat carcasses. For this study eight female rat carcasses were placed in plastic boxes, with plastic nets on the sites to allow insects to enter, on the roof of the Forensic Institute in France. Before the rats were placed in the boxes, six of them were treated with different substances: unleaded petroleum, patchouli perfume, hydrochloric acid (HCl), insecticide spray, sodium hydroxide and mosquito citronella. Two rats were used as control. At different moments during one month the present insects were collected, killed and identified. This experiment

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20 was repeated for four consecutive years, in the same period. Also, olfactometer experiments were performed using C. Vicina and mouse cadavers to confirm the field experiments in controlled conditions. Therefore, the flies were placed in a transparent tube with an air pump creating an odour gradient through the tube, and the walk behaviour of the flies was studied. The field experiments showed significant repellent effects of HCl, patchouli perfume, insecticide and gas compared to the other substances and the control samples (Table 1). HCl and patchouli perfume showed the strongest effect, delaying the insect colonization with 73 hours and 101 hours respectively. The olfactometer experiments also showed a repellent effect of gas, insecticide and HCl, but also of mosquito citronella and paradichlorobenzene, a substance not tested in the field experiments. Patchouli perfume was not tested in the olfactometer experiments. This study shows that various household products can be used to alter and reduce the smell produced by cadavers and the subsequent attraction of Diptera towards carcasses.

During an investigation at the Anthropology Research Facility (ARF) in Tennessee the hand of a female body was removed and treated with formalin to take fingerprints, after which the hand was put back with the body. After months of outdoor exposure, no maggots appeared to feed on the hand, although the rest of the body was in advanced state of decay (Mann et al., 1990). This implicates a reduced attractiveness of Diptera towards tissue treated with formalin. Vass (2001) also mentioned a case in which a human body showed low insect activity due to a chemical treatment. A specific case was described of a dead woman found outside, showing almost no signs of decomposition and insect activity. Investigators believed that the woman died quite recently, but eventually the woman appeared to be dead for four months already. After death she was spread with insecticide by the murderer to mask the odour. Although some internal decomposition had taken place, the insecticide had prevented insect attraction and oviposition, strongly inhibiting the decomposition process.

These examples show that the use of chemicals on a body can significantly inhibit the attraction of Diptera towards human carcasses, which subsequently leads to slower tissue decomposition. It is therefore important to investigate the presence of chemicals on found corpses, before PMI estimations with entomological methods are done. However, it is remarkable that during the research of Charabidze

Table 1 | Summary of field and walk olfactometer results. An * indicates a significant repellent effect (α=0.005).

Modified from Charabidze et al. (2009).

Field experiments Olfactometer experiments

Patchouli perfume Repulsive* Not tested

Paradichlorobenzene Not tested Repulsive*

Gas Repulsive* Repulsive*

Insecticide Repulsive* Repulsive*

Hydrochloric acid Repulsive* Repulsive*

Mosquito citronella repellent No effect Repulsive*

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et al. (2009), into the effect of different household products on fly attraction, the field experiments showed partly different results than the laboratory (olfactometer) experiments. Mosquito citronella repellent showed a significant repellent effect in the olfactometer experiments but did not show an effect during the field experiments. The reason for this difference remains unclear.

5.4 Intrinsic factors

Besides the external factors that can be applied to a body, such as physical hiding or treatment with chemical substances, also intrinsic characteristics of the body itself can have an impact on the volatiles produced and subsequent fly attraction. One of these intrinsic characteristics is the size of the body.

5.4.1 Body weight

Hewadikaram and Goff (1991) compared the process of decomposition and insect succession of two pig carcasses, weighing 8.4 kg and 15.1 kg. The cadavers were placed outside on a study site in Hawaii and checked twice a day in the first week, once a day until day 23 and three times a day after that. During every check the carcasses were weighed, arthropod activity was observed, and specimens were collected for identification. This study showed that the patterns of decomposition were similar for both pig cadavers, but that the rate of decomposition, especially during decay and post-decay stages (day 5-16), was higher for the 15.1 kg carcass relative to the 8.4 kg carcass. This corresponds to the higher number of adult Diptera that were attracted towards the bigger cadaver, and associated greater number of fly larvae, responsible for rapid removal of biomass. However, the pattern of insect succession and temporal occurrence and development of the flies were similar. This indicates that the volatiles produced by both carcasses were detected by flies at the same time, but that the higher biomass and associated higher level of produced volatiles of the 15.1 kg cadaver attracted relatively more flies.

Campobasso et al. (2001) suggested that obese human cadavers contain higher amounts of liquid, which favours the development and spread of bacteria. This could lead to an earlier onset of active decay, and corresponding earlier spreading of volatile substances and fly detection. Also Mann et al. (1990) suggest that body fats in obese bodies are quickly degraded, during which fluids escape from the body. This could induce earlier attraction by Diptera. However, no study has confirmed this yet.

5.4.2 Medication, drugs and alcohol

Also, the presence of drugs, medication, poison or alcohol inside a body can affect the attraction of flies towards a carcass. When looking into the presence of these substances in decomposing bodies, a relatively new topic within forensic entomology comes into play: entomotoxicology. Entomotoxicology is the study of the effects of drugs (in human cadavers) on insect development, to gain more insight in the effects of drugs on PMI estimations using insects. Also, insects can be used to detect drugs and/or toxins in decomposing cadavers, as insects and larvae feed on soft tissues of decomposing bodies to grow and develop (Introna et al., 2001). Some research is done into the effects of different substances on insect development.

Zou et al. (2013) studied the effect of different concentrations of ketamine in liver and muscle tissue of ten rabbit cadavers on growth and development of Lucilia sericata (Calliphoridae). This study shows that ketamine accelerated the growth of this fly species. Also cocaine appears to induce higher

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22 grow rates of Calliphoridae (C. albiceps and C. putoria), showed by De Carvalho et al. (2012). In this study, livers of ten rabbit cadavers, eight of which treated with cocaine before death and two as control, were used as feeding site for the two fly species. The weight of the larvae present on cocaine treated livers increased significantly faster than the larvae on control livers. For C. albiceps, 54 hours after exposure larvae present on cocaine treated livers weighed 1100 mg compared to 600 mg weighing larvae present on control livers. At the same time point larvae of C. putoria weighed 500 mg on cocaine treated livers compared to 200 mg weighing larvae present at control livers. Besides the effect of cocaine on growth rate, the larvae present on cocaine treated livers also pupated significantly earlier than the larvae present on the control livers. In contrast to ketamine and cocaine, George et al. (2009) suggest that morphine does not significantly affect the growth rate of Calliphoridae (C. stygia). For this study fly larvae were placed on pet minced treated with different concentrations of morphine and a control. At different time points during larvae development length and width were measured. This study shows no growth differences between larvae present on morphine treated meat compared to larvae growing on meat not containing morphine.

These studies show that different types of drugs can affect the development of Calliphoridae larvae in different ways. When looking into the effect of different unnatural substances inside corpses on insect attraction, less research is done yet. AbouZied (2016) studied the effect of tramadol, a synthetic opioid used as pain medication, on the decomposition process of rat carcasses and the attraction of different Diptera species towards the carcasses. For this study twenty laboratory rats, ten of which treated with tramadol and ten of which used as control, were used to investigate the effect of tramadol on decomposition. Therefore, carcasses were placed outside four to six days after death and observed daily for one month. The same experiments were repeated, with in total ten rats, and larvae were collected at the end of the bloated stage, killed and identified. During the field experiments, three fly traps were placed close to tramadol-treated rat carcasses and three close to control rat carcasses. Trapped flies were collected daily, killed, counted and identified. This study showed that the fresh stage of tramadol-treated carcasses was significantly shorter than for the control carcasses; 2.4 (±0.27) days compared to 6.4 (±0.49) days. The dry stage, however, showed to be significantly longer for the tramadol-treated rats compared to the control rats; 10.3 (±0.99) days compared to 7.4 (± 0.18) days. The overall duration of decomposition of tramadol-treated and control rats did not differ significantly. In the fly traps different fly species (such as C. albiceps, L. cuprina, Sarcopahaga sp. and M. domestica) were found. L. cuprina, C. albiceps and M. domestica were less found in traps around tramadol-treated carcasses compared to traps around control rats. For L. cuprina an average of 13 flies a day were found around tramadol-treated carcasses compared to 0 flies around control carcasses. The results for C. albiceps and M. domestica were 26 to 6 and 5 to 0 respectively. However, the total number of Sarcophagidae (Sarcophaga sp. and Wohlfartia sp.) trapped around tramadol-treated rats was higher compared to the number of flies trapped around control rats. This study suggests that the attraction of flies towards rat carcasses can be increased or inhibited by the treatment with tramadol, dependent on the fly species. These results suggest that, although often is assumed that all fly species are attracted towards corpses in a comparable manner, different species can show significant different carcass detection and attraction.

Factors affecting the attraction of Diptera towards human carcasses - a literature study