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      Characterizing the scent and chemical composition of Panthera leo marking fluid using solid-phase microextraction and multidimensional gas chromatography–mass spectrometry-olfactometry

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      Scientific Reports
      Nature Publishing Group UK

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          Abstract

          Lions ( Panthera leo) use chemical signaling to indicate health, reproductive status, and territorial ownership. To date, no study has reported on both scent and composition of marking fluid (MF) from P. leo. The objectives of this study were to: 1) develop a novel method for simultaneous chemical and scent identification of lion MF in its totality (urine + MF), 2) identify characteristic odorants responsible for the overall scent of MF as perceived by human panelists, and 3) compare the existing library of known odorous compounds characterized as eliciting behaviors in animals in order to understand potential functionality in lion behavior. Solid-phase microextraction and simultaneous chemical-sensory analyses with multidimensional gas-chromatography-mass spectrometry-olfactometry improved separating, isolating, and identifying mixed (MF, urine) compounds versus solvent-based extraction and chemical analyses. 2,5-Dimethylpyrazine, 4-methylphenol, and 3-methylcyclopentanone were isolated and identified as the compounds responsible for the characteristic odor of lion MF. Twenty-eight volatile organic compounds (VOCs) emitted from MF were identified, adding a new list of compounds previously unidentified in lion urine. New chemicals were identified in nine compound groups: ketones, aldehydes, amines, alcohols, aromatics, sulfur-containing compounds, phenyls, phenols, and volatile fatty acids. Twenty-three VOCs are known semiochemicals that are implicated in attraction, reproduction, and alarm-signaling behaviors in other species.

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          Most cited references115

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          Sophisticated sperm allocation in male fowl.

          When a female is sexually promiscuous, the ejaculates of different males compete for the fertilization of her eggs; the more sperm a male inseminates into a female, the more likely he is to fertilize her eggs. Because sperm production is limited and costly, theory predicts that males will strategically allocate sperm (1) according to female promiscuity, (2) saving some for copulations with new females, and (3) to females producing more and/or better offspring. Whether males allocate sperm in all of these ways is not known, particularly in birds where the collection of natural ejaculates only recently became possible. Here we demonstrate male sperm allocation of unprecedented sophistication in the fowl Gallus gallus. Males show status-dependent sperm investment in females according to the level of female promiscuity; they progressively reduce sperm investment in a particular female but, on encountering a new female, instantaneously increase their sperm investment; and they preferentially allocate sperm to females with large sexual ornaments signalling superior maternal investment. Our results indicate that female promiscuity leads to the evolution of sophisticated male sexual behaviour.
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            Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors.

            The fruit fly, Drosophila melanogaster Meigen (Diptera: Drosophilidae), is a model for how animals sense, discriminate, and respond to chemical signals. However, with D. melanogaster our knowledge of the behavioral activity of olfactory receptor ligands has relied largely on close-range attraction, rather than on long-range orientation behavior. We developed a flight assay to relate chemosensory perception to behavior. Headspace volatiles from vinegar attracted 62% of assayed flies during a 15-min experimental period. Flies responded irrespective of age, sex, and mating state, provided they had been starved. To identify behaviorally relevant chemicals from vinegar, we compared the responses to vinegar and synthetic chemicals. Stimuli were applied by a piezoelectric sprayer at known and constant release rates. Re-vaporized methanol extracts of Super Q-trapped vinegar volatiles attracted as many flies as vinegar. The main volatile component of vinegar, acetic acid, elicited significant attraction as a single compound. Two other vinegar volatiles, 2-phenyl ethanol and acetoin, produced a synergistic effect when added to acetic acid. Geosmin, a microbiological off-flavor, diminished attraction to vinegar. This wind tunnel assay based on a conspicuous and unambiguous behavioral response provides the necessary resolution for the investigation of physiologically and ecologically relevant odors and will become an essential tool for the functional analysis of the D. melanogaster olfactory system.
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              Gas chromatography-olfactometry.

              GC-olfactometry (GC-O) refers to the use of human assessors as a sensitive and selective detector for odour-active compounds. The aim of this technique is to determine the odour activity of volatile compounds in a sample extract, and assign a relative importance to each compound. Methods can be classified into three types: detection frequency, dilution to threshold and direct intensity. Dilution to threshold methods measure the potency of odour-active compounds by using a series of extract dilutions, whereas detection frequency and direct-intensity methods measure odour-active compound intensity, or relative importance, in a single concentrated extract. Factors that should be considered to improve the value of GC-O analysis are the extraction method, GC instrument conditions, including the design and operation of the odour port, methods of recording GC-O data and controlling the potential for human assessor bias using experimental design and a trained panel. Considerable emphasis is placed on the requirement for multidimensional GC analysis, and on best practice when using human assessors.
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                Author and article information

                Contributors
                koziel@iastate.edu
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                11 July 2017
                11 July 2017
                2017
                : 7
                : 5137
                Affiliations
                [1 ]ISNI 0000 0004 1936 7312, GRID grid.34421.30, , Iowa State University, Environmental Science Graduate Program, ; Ames, IA 50011 United States of America
                [2 ]ISNI 0000 0004 1936 7312, GRID grid.34421.30, , Iowa State University, Department of Agricultural and Biosystems Engineering, ; Ames, IA 50011 United States of America
                Author information
                http://orcid.org/0000-0002-2387-0354
                Article
                4973
                10.1038/s41598-017-04973-2
                5506057
                28698649
                5be0abe7-67d5-459a-b1c3-1c96545872eb
                © The Author(s) 2017

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 October 2016
                : 22 May 2017
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