Size matters: Natural experiments suggest the dear enemy effect is moderated by pack size in African wild dogs.

Remote monitoring of communal marking sites, or latrines, provides a unique opportunity to observe undisturbed scent marking behaviour of African wild dogs (Lycaon pictus). We used remote camera trap observations in a natural experiment to test behavioural scent mark responses to rivals (either familiar neighbours or unfamiliar strangers), to determine whether wild dogs exhibit the "dear enemy" or "nasty neighbour" response. Given that larger groups of wild dogs represent a greater threat to smaller groups, including for established residents, we predicted that the overarching categories "dear enemy" vs. "nasty neighbour" may be confounded by varying social statuses that exists between individual dyads interacting. Using the number of overmarks as a metric, results revealed an interaction between sender and receiver group size irrespective of familiarity consistent with this prediction: in general, individuals from large resident packs overmarked large groups more than they overmarked smaller groups, whereas individuals from smaller packs avoided overmarking larger groups, possibly to avoid detection. Monitoring a natural system highlights variables such as pack size that may be either overlooked or controlled during scent presentation experiments, influencing our ability to gain insights into the factors determining territorial responses to rivals.

Chemical signals in terrestrial vertebrates: search for design features.

We compiled a data set of the compounds that terrestrial vertebrates (amniotes) use to send chemical signals, and searched for relationships between signal compound properties and signal function. Overall, relationships were scarce and formed only small-scale patterns. Terrestrial vertebrate signalling compounds are invariably components of complex mixtures of compounds with diverse molecular weights and functionalities. Signal compounds with high molecular weights (MWs) and low vapour pressures, or that are bound to carrier proteins, are detected during direct contact with the source of the signal. Stable compounds with aromatic rings in their structures are more common in signals of social dominance, including territoriality. Aldehydes are emitted from the sender's body rather than from scent marks. Lipocalin pheromones and carriers have a limited range of MWs, possibly to reduce the metabolic costs of their biosynthesis. Design constraints that might channel signal chemistry into patterns have been relaxed by amniote behavior and biochemistry. Amniote olfaction has such a high sensitivity, wide range and narrow resolution that signal detection imposes no practical constraints on the structures of signalling molecules. Diverse metabolic pathways in amniotes and their microbial commensals produce a wide variety of compounds as chemical signals and as matrix compounds that free signal components from the constraints of stability, vapor pressure, species-specificity etc. that would otherwise constrain what types of compound operate optimally under different conditions.

Are mammal olfactory signals hiding right under our noses?

Chemical communication via olfactory semiochemicals plays a central role in the social behaviour and reproduction of mammals, but even after four decades of research, only a few mammal semiochemicals have been chemically characterized. Expectations that mammal chemical signals are coded by quantitative relationships among multiple components have persisted since the earliest studies of mammal semiochemistry, and continue to direct research strategies. Nonetheless, the chemistry of mammal excretions and secretions and the characteristics of those semiochemicals that have been identified show that mammal semiochemicals are as likely to be single compounds as to be mixtures, and are as likely to be coded by the presence and absence of chemical compounds as by their quantities. There is very scant support for the view that mammal semiochemicals code signals as specific ratios between components, and no evidence that they depend on a Gestalt or a chemical image. Of 31 semiochemicals whose chemical composition is known, 15 have a single component and 16 are coded by presence/absence, one may depend on a ratio between two compounds and none of them are chemical images. The expectation that mammal chemical signals have multiple components underpins the use of multivariate statistical analyses of chromatographic data, but the ways in which multivariate statistics are commonly used to search for active mixtures leads to single messenger compounds and signals that are sent by the presence and absence of compounds being overlooked. Research on mammal semiochemicals needs to accommodate the possibility that simple qualitative differences are no less likely than complex quantitative differences to encode chemical signals.

Identification of volatiles from the secretions and excretions of African wild dogs (Lycaon pictus).

Gas chromatography/mass spectrometry was used to identify 103 organic compounds from urine, feces, anal glands, and preputial glands of free-ranging African wild dogs, Lycaon pictus. Aliphatic acids were the dominant class of compound in all materials. In addition to aliphatic acids, urine contained dimethyl sulfone, 1,3-propanediol, benzoic acid, 1-methyl-2,4-imidazolidinedione, and squalene as major components: feces contained indole and cholesterol; and both contained 2-piperidone, phenol, 4-methyl phenol, benzeneacetic acid, and benzenepropanoic acid and other compounds. Anal gland secretion was particularly rich in cholesterol and fatty acids, and preputial gland secretion rich in squalene. A large majority of the identified compounds have been reported from other mammals, including species sympatric with African wild dogs. Eleven of the African wild dog components have not been reported previously from mammals and have not been found in sympatric species; one component, 1-methylimidazole-5-carboxaldehyde has not been reported previously as a natural product. In the chemical profiles of their urine, feces, and anal gland secretion African wild dogs differ markedly from other canids.

A versatile cryo-focussing flow switching gas chromatography inlet for trace analysis of intractable compounds.

A wide variety of samples that can be analysed by gas chromatography do not lend themselves to the usual preparation of solvent extracts for split-splitless injections, and are best handled by purge and trap or equilibrium headspace sampling. A cryo-focussing, flow switching gas chromatography inlet system that handles different types of sample without the need for hardware changes has been prototyped. It provides excellent repeatability and linearity with liquid injections, purge and trap, and equilibrium headspace samples, in both split and splitless modes. The performance of the system was tested with sub-nanogram quantities of challenging analytes such as free carboxylic acids, alcohols, diols, phenols and aldehydes, and volatiles purged from contaminated soil, mammal faeces, a pesticide formulation, and a spice. Repeatability RSDs for peak areas were consistently below 11% and repeatabilities of retention times below 0.05%, independently of sample type (liquid or gas phase) and nature or quantity of compound. Regression coefficients of peak areas vs. quantity were typically ≥ 0.999 over two orders of magnitude ranges extending down to below 0.01 ng, also independently of sample and analyte. Limits of quantitation were robustly below 0.1-0.2 ng. Peak shapes and resolution are the same with use of the cryo-trap and flow switch as they are with conventional injections. Performance is robust to flow rate and, for most compounds, to trapping and desorption temperature. The cryo-trapping flow switching inlet's performance parameters match those of other sample introduction systems, and are achieved with sub-nanogram quantities of intractable analytes.

Quantification of appetite suppressing steroid glycosides from Hoodia gordonii in dried plant material, purified extracts and food products using HPLC-UV and HPLC-MS methods.

High-performance liquid chromatography (HPLC)-UV and HPLC-Mass Spectrometry (MS) methods were developed for the quantitative analysis of the family of Hoodia gordonii steroid glycosides with appetite suppressing properties in dried plant material, in purified and enriched extracts and in various prototype food-products fortified with H. gordonii extracts. For solid materials, e.g. dried plants or for non-fatty foods, extraction of the steroid glycosides is performed using methanol. For products where the steroid glycosides are present in an oil matrix, direct injection of the oil after dilution in tetrahydrofuran is applied. The HPLC separation is performed on an octyl-modified reversed-phase column in the gradient mode with UV detection at lambda = 220 nm. Quantification is performed against an external calibration line prepared using either one of the pure steroid glycosides or geranyl-tiglate. Short- and long-term repeatabilities of the methods are better than 3 and 6%, respectively. Recoveries are better than 85%, even in the analysis of the least abundant steroid glycosides in a complex yoghurt drink. Linearity is better than 3-4 orders of magnitude and the detection limits are below approximately 2 microg g(-1) for the individual steroid glycosides in dried plant material and food products. HPLC-MS is used to confirm that the steroid glycosides contain the characteristic steroid core, the carbohydrate chain and the tigloyl group.

Low cost, robust, in-house hardware for heart cutting two-dimensional gas chromatography.

Natural materials are so complex that no single column can separate all of the components. Heart cutting 2-D GC (GC-GC) using a Deans switch provides maximum separation, but the requisite gas flow configurations have earned a reputation for being fiddly and time consuming to set up and tune, unless electronic gas controls and costly software are employed. A design for a vented Deans switch is presented that can be assembled in-house from standard commercial components. A vent to atmosphere replaces the balancing resistor, which solves the problem of balancing the pressures and flows, and requires no compensation for changes in gas viscosity and back-pressure during temperature programming. First and second dimension columns can both be run at optimum flow rates, even if they are of different diameters. Analyte detectability is preserved by cutting only the target fraction from the first column and transferring the whole of it to the second column. Cryotrapping the selected fraction allows cuts of any width, and transfer of analytes to the second column as sharp bands. I have applied the vented Deans switch-cold trap to the identification of flavour and fragrance compounds from South African plants and foods; cuts are very repeatable, and the detectability of trace components is enhanced. Its capabilities are demonstrated by examples from analyses of essential oils and flavour extracts.

Enhanced flavour extraction in continuous liquid-liquid extractors.

Continuous liquid-liquid extraction is a versatile, reliable and robust sample preparation technique, but there is a tendency for the solvent to make channels through the sample, causing uneven and incomplete extraction. A simple, cheap magnetic stirrer that prevents channelling improves extraction efficiency by between 2.8 and 17.6 times.