Chemical Ecology Research

The identification of semiochemicals and their purpose can be achieved by examining the odour source and understanding their role in the biology of both the insect and the host. Various chemical ecology techniques can facilitate this. Volatile semiochem-icals can be isolated using air entrainment (also referred to as headspace collection). This method allows the collection of volatile compounds produced by an odour source (e.g. decomposing body) onto a filter, commonly comprising a porous polymer, such as Porapak or Tenax. During this process contamination is kept to a minimum by, isolating the target source, with only the volatile chemicals produced by the source being collected (Agelopoulos et al. 1999). Subsequent analytical chemistry techniques, such as gas chromatography (GC) and GC-mass spectrometry (GC-MS) allow the chemicals to be identified accurately.

Headspace analysis is an established technique already used in forensic science for the collection of volatiles in blood and organ specimens (Statheropoulos et al. 2005; Hoffman et al. 2009). However, it has also been used successfully to investigate volatile odours from dead bodies. For example, Statheropoulos et al. (2005) used air entrainment to collect volatiles from the bodies of two males and identified and quantified over 80 different chemicals. The most prominent compounds were dimethyl disulfide, toluene, hexane, benzene 1,2,4-trimethyl, 2-propanone and 3-pentanone and they found marked differences in concentration between the two bodies. The authors suggest that these differences could reflect different rates of decomposition between the bodies and thus may provide valuable information about time of death (Statheropoulos et al. 2005). Later Statheropoulos et al. (2007) collected air entrainment samples of a body during the early stages of decomposition (4 days since death) during a period of 24 h. This time, over 30 volatile chemicals were identified. Eleven of these compounds were recovered throughout each sample, forming a "common core". The "common core" was made-up of the following: etha-nol, 2-propanone, dimethyl disulfide, methyl benzene, octane, o-xylene, m-xylene, p-xylene, 2-butanone, methyl ethyl disulfide and dimethyl trisulfide (Statheropoulos et al. 2007). More recently, a study was conducted to identify volatile compounds from 14 separate tissue samples (Hoffman et al. 2009). In total 33 compounds were identified and could be grouped into seven chemical classes such as alcohols, acid esters, aldehydes, halogens, aromatic hydrocarbons, ketones and sulfides. There were common compounds identified in all of these studies; however, it was found that, at this stage, no unique set of compounds could be used to create a "chemical signature" of the decomposing tissues.

Similarly, volatiles have been collected successfully from dead pigs (sus scrofa). In this case the pigs were contained inside a metal container during the time of sampling and the volatiles were extracted through Porapak and Tenax filters (LeBlanc 2008). These samples were taken daily in order to encompass the different stages of decomposition. A large number compounds were recovered, however, in this research the aim was to locate specific compounds which are detected carrion insects, specifically the blowfly Calliphora vomitoria (Diptera: Calliphoridae).

Although air entrainment followed by GC and GC-MS analysis can provide information regarding the general profile from an odour source (often hundreds of volatile chemicals), this alone does not indicate which volatiles are detected by the carrion insects antennae (i.e. those that are electrophysiologically-active). Therefore, additional techniques can be used to discriminate between electrophysiological^ active and inactive compounds in the complex extract (Wadhams 1990). By combining different analysis methods, the identification of volatile compounds can be refined to those which have a behavioural impact on insects associated with decomposing bodies and thus, those that are characteristic of a particular stage of decomposition.

Electroantennogram (EAG) recordings were originally utilised by Schneider in 1957. Using microelectrodes, he found that it was possible to record depolarisation of the affected sensillum on the antenna stimulated by a volatile compound introduced over the antennal preparation (Fig. 11.3). In the case of Dipterans such as Muscidae or Calliphoridae, the antennae are connected to microelectrodes that record the response of the olfactory receptor neurones in the antennae. An odour stimulus can be delivered through an air stream flowing continuously over the preparation and a response (depolarisation) can be immediately recorded if the compound elicits an electrophysiological response. This is an effective means of initially identifying semiochemicals because EAG responses are recorded without the influence of environmental or neurological factors which could affect behavioural responses (Cork et al. 1990). EAG can be combined with gas chromatography to give GC-EAG which allows the location of active chemicals within a complex extract. This technique allows the location of EAG-active chemicals within complex extracts by taking advantage of the high-resolution of the GC while simultaneously utilising the

Provided by Images of Nature

Fig. 11.3 Scanning electron microscope (SEM) image of Muscidae

Provided by Images of Nature

Fig. 11.3 Scanning electron microscope (SEM) image of Muscidae extreme sensitivity and selectivity of the antennal preparation of an insect. A volatile sample is injected into the GC and the sample is split in two - half of the sample travels to the flame ionisation detector (FID) and half is simultaneously passed over the insect preparation (Fig. 11.4). The compound is recorded and displayed within a chromatogram and the reaction from the fly, if any, is recorded as a depolarisation indicated along with the compound in the chromatogram (Fig. 11.5). GC-EAG was first reported in 1969 (Moorhouse et al. 1969) and was subsequently advanced to

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