http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0003591 Genetically-Based Olfactory Signatures Persist Despite Dietary Variation Jae Kwak1, Alan Willse2B$a, Koichi Matsumura1, Maryanne Curran Opiekun1, Weiguang Yi1B$b, George Preti1,3, Kunio Yamazaki1, Gary K. Beauchamp1* 1 Monell Chemical Senses Center, Philadelphia, Pennsylvania, United States of America, 2 Battelle - Pacific Northwest Division, Richland, Washington, United States of America, 3 Department of Dermatology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America Abstract Individual mice have a unique odor, or odortype, that facilitates individual recognition. Odortypes, like other phenotypes, can be influenced by genetic and environmental variation. The genetic influence derives in part from genes of the major histocompatibility complex (MHC). A major environmental influence is diet, which could obscure the genetic contribution to odortype. Because odortype stability is a prerequisite for individual recognition under normal behavioral conditions, we investigated whether MHC-determined urinary odortypes of inbred mice can be identified in the face of large diet-induced variation. Mice trained to discriminate urines from panels of mice that differed both in diet and MHC type found the diet odor more salient in generalization trials. Nevertheless, when mice were trained to discriminate mice with only MHC differences (but on the same diet), they recognized the MHC difference when tested with urines from mice on a different diet. This indicates that MHC odor profiles remain despite large dietary variation. Chemical analyses of urinary volatile organic compounds (VOCs) extracted by solid phase microextraction (SPME) and analyzed by gas chromatography/mass spectrometry (GC/MS) are consistent with this inference. Although diet influenced VOC variation more than MHC, with algorithmic training (supervised classification) MHC types could be accurately discriminated across different diets. Thus, although there are clear diet effects on urinary volatile profiles, they do not obscure MHC effects. Citation: Kwak J, Willse A, Matsumura K, Curran Opiekun M, Yi W, et al. (2008) Genetically-Based Olfactory Signatures Persist Despite Dietary Variation. PLoS ONE 3(10): e3591. doi:10.1371/journal.pone.0003591 Editor: Hiroaki Matsunami, Duke Unviersity, United States of America Received: April 21, 2008; Accepted: September 26, 2008; Published: October 31, 2008 Copyright: B) 2008 Kwak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by ARO contract DAAD19-03-1-0109. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the United States Government. Competing interests: The authors have declared that no competing interests exist. * E-mail: beauchamp@monell.org B$a Current address: Monsanto Company, Ankeny, Iowa, United States of America B$b Current address: Department of Horticulture, University of Georgia, Athens, Georgia, United States of America Introduction For social animals, an ability to identify individuals is almost a prerequisite for the efficient organization of behavioral interactions. Many studies have demonstrated that individual animals can recognize one another as individuals and that, for mammals in particular, body volatiles (herein referred to loosely as odors) detected by olfactory or vomeronasal receptors play a prominent role in mediating this individual recognition [1]. We have previously proposed that each individual mouse has a unique odor which we have termed its odortype [2]. Odortypes, like other phenotypes, are influenced by genetic and environmental variation, and possibly their interaction. Among the genetic bases for individual odortypes, variation in genes of the MHC plays a central role as we and many others have demonstrated [2]b[5]. Variation in non-MHC genes can also influence the urinary odors of mice and contribute toward specification of genetically-determined odortypes [6]b[8]. Individual identity, as it is commonly conceived, is the sum of the characteristics of an individual animal that distinguish it from other members of its species. It is generally assumed that these individual characteristics must be relatively stable over considerable time periods so that the individual can be recognized in multiple behavioral and social contexts. Thus for odortypes it would seem desirable that they be stable over time and relatively uninfluenced by day-to-day fluctuations due to such factors as variation in diet. Nevertheless, there is considerable evidence suggesting short-term fluctuations in body odors due to variation in stress [9], disease state [10], [11] and diet [12], [13 and reference therein]. Some reports suggest that dietary changes might mask genetically determined odortypes, preventing individual recognition [14], [15]. However, it would be surprising if an altered diet made it impossible to recognize genetically-determined individuality of odor, as Schellinck et al [15] suggest. Instead, we hypothesize that genetically-determined odortypes, and particularly MHC-determined odortypes, are relatively buffered against changes due to short-term environmental fluctuations. Such buffering would seem to be a prerequisite for MHC odortypes to be involved in mediating mate choice in natural environments, as studies in semi-natural testing conditions suggest [16]. To clarify the influence of diet on MHC-regulated odortypes, we conducted combined behavioral and chemical studies using urine samples from two different congenic mouse strains each on two different diets. First, we tested whether MHC odortypes are perceived following substantial changes in diet. We found that although diet clearly has a large effect on urinary odors, MHC-determined odortype variation can be recognized in spite of major diet variation. Chemical analyses of urinary VOCs for these same mice were completely consistent with behavioral results: dietary variation significantly altered the profile of urinary VOCs, but a clear subset of MHC determined VOCs was unperturbed by diet variation, allowing for statistical discrimination of MHC types across dietary treatments. ...