Bacteria isolated from bengal cat (Felis catus × Prionailurus bengalensis) anal sac secretions produce volatile compounds associated with animal signaling

Anal sacs are an important odor producing organ found across the mammalian Order Carnivora. Secretions from the anal sac may be used as chemical signals by animals for behaviors ranging from defense to species recognition to signaling reproductive status. In addition, a recent study suggests that domestic cats utilize short-chain free fatty acids in anal sac secretions for individual recognition. The fermentation hypothesis is the idea that symbiotic microorganisms living in association with animals contribute to odor profiles used in chemical communication and that variation in these chemical signals reflects variation in the microbial community. Here we examine the fermentation hypothesis by characterizing volatile organic compounds (VOC) and bacteria isolated from anal sac secretions collected from a male bengal cat, a cross between a domestic cat and wild leopard cat (Felis catus × Prionailurus bengalensis). Both left and right anal sacs of a male bengal cat were manually expressed (emptied) and collected. Half of the material was used to culture bacteria or to extract bacterial DNA and other half was used for VOC analysis. DNA was extracted from the anal sac secretions and used for a 16S rRNA gene sequence based characterization of the microbial community. Additionally, some of the material was plated out in order to isolate bacterial colonies. The same three taxa, Bacteroides fragilis, Tessaracoccus, and Finegoldia magna were abundant in the 16S rRNA gene sequence data and also isolated by culturing. Using Solid Phase Microextraction (SPME) gas chromatography-mass spectrometry (GC-MS), we tentatively identified 52 compounds from bengal cat anal sac secretions and 67 compounds from cultures of the three bacterial isolates chosen for further analysis.. Among 67 compounds tentatively identified from bacteria isolates, 52 were also found in the anal sac secretion. We show that the bacterial community in the anal sac consists primarily of only a few abundant taxa and that isolates of these taxa produce numerous volatiles that are found in the combined anal sac volatile profile. Many of these volatiles are found in anal sac secretions from other carnivorans, and are also associated with known bacterial biosynthesis pathways. This supports the fermentation hypothesis and the idea that the anal sac is maintained at least in part to house bacteria that produce volatiles for the host.

The chemical composition of anal sac secretions has been analyzed in a number of animals in the 68 Carnivora. Studies in the cheetah (Acinonyx jubatus), red fox (Vulpes vulpes), dog (Canis familiaris), 69 coyote (Canis latrans), wolf (C. lupus), lion (Panthera leo), and mongoose (H. auropunctatus) have 70 identified volatile short-chain free fatty acids, such as acetic acid, propanoic acid, and butanoic acid as 71 being partially responsible for the odors [22][23][24][25][26][27][28]. The nature of these constituents led to the suggestion that 72 they may be metabolites produced by bacteria in the sac from available substrates [22]. 73 The fermentation hypothesis posits that bacteria metabolize secretions and produce volatile organic 74 compounds, such as hydrocarbons, fatty acids, wax esters, and sulfur compounds [15,16,29] that are used 75 in communication by the host [30,31]. Evidence in support of this hypothesis links bacterial action to 76 specific, olfactory-mediated host behavior or to the production of certain odorants. For example, researchers 77 have shown that trimethylamine, an odorant that plays a key role in mouse (Mus musculus) reproduction, 78 requires commensal bacteria for its production [24]. Researchers have also inhibited odorant production in 79 Indian mongooses (H. auropunctatus) and European hoopoes (Upupa epops) by treating the animals' scent 80 glands with antibiotics [30,32].

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In this study, we investigated the fermentation hypothesis by focusing in detail on bacterial isolates 82 collected from a single animal, available at the time of our study. We studied a bengal cat, a hybrid between 83 the Asian leopard cat (P. bengalensis) and the domestic cat (F. catus). We collected anal sac secretions in 84 order to characterize their chemical profile and analyze bacterial community composition. Then we isolated 85 and identified those bacteria that could be cultivated under anaerobic conditions from these samples. 86 Volatiles produced by these isolates were identified and compared to those found in the anal sac secretions. 87 This is the first study in felines to demonstrate that bacteria isolated from anal sacs produce key volatile 88 compounds found in anal sac secretions.  Raw, demultiplexed amplicon reads were processed using DADA2 v1.8, following the standard 119 online tutorial [34]. The reads were trimmed down to 250 base pairs to remove low quality nucleotides. In 120 addition, the quality of reads were ensured by trimming bases that did not satisfy a Q2 quality score. Reads 121 containing Ns were discarded and we used two expected errors to filter the overall quality of the read (rather 122 than averaging quality scores) [35]. Chimeric reads were also removed using DADA2 on a per sample 123 basis. The remaining pairs of reads were merged into amplicon fragments and unique Amplicon Sequence 124 Variants (ASVs) were identified. Reads that did not merge successfully were discarded. Upon completion 125 of the DADA2 pipeline, all ASVs (n=52) that were found in the negative control swabs were removed from 126 the analysis, only three of these ASVs were also found in the anal sac samples. ASVs were assigned 127 taxonomy using the dada2 function "assignTaxonomy" and the Silva (NR v132) database [36-38]. One 128 ASV that was assigned to "Eukaryotes" was removed. All ASVs with the same taxonomy (at the genus 129 level) were grouped and then ranked by number of reads. No ASVs were assigned to mitochondria or 130 chloroplast. 131 132 Bacterial culturing and identification 133 Anal sac secretions were vortexed with 1 mL Phosphate Buffer Saline (PBS). Two serial 1:10 134 dilutions were performed and 100 µL of each dilution was plated onto Columbia Blood Agar (CBA) and 135 Brain Heart Infusion (BHI). Plates were incubated anaerobically in a BD GasPak EZ Container System 136 with packets of CO 2 generator for 5 days at 37 °C. Morphologically distinct colonies were streaked for 137 isolation on both CBA and BHI. The 16S rRNA gene was sequenced using Sanger sequencing using the 138 27F and 1391R primers. Taxonomy was assigned by the result of BLAST queries to the nr database at 139 NCBI (excluding unnamed/environmental sequences), a species name was given in cases where the identity 140 was >98% to only a single species. 141 142 Bacterial volatile analysis 143 To extract volatiles from Bacteroides fragilis UCD-AAL1 and Tessaracoccus sp. UCD-MLA, 144 cultures were grown in 5 mL BHI anaerobically for 24 hours at 37 °C. 100 µL of the culture was transferred 145 into each of three Restek (Bellefonte, PA) tubes filled with 5 mL of BHI. Two jar blanks (no media or 146 bacteria) and two BHI media-only blanks were used as controls. The same procedure was followed for 147 Finegoldia magna UCD-MLG, except that cultures were grown and incubated in BHI supplemented with 148 5% defibrinated sheep blood (BBHI) anaerobically for 24 hours at 37°C. 149 Headspace extraction was performed with Solid Phase Microextraction (SPME) fibers (Part 57912-150 U, Sigma Aldrich) which had 50/30 μm thickness and DVB/CAR/PDMA coating. Two SPMEs were 151 inserted into the headspace of each Restek tube prior to anaerobic incubation at 37 °C for 24 hours. SPME 152 fibers were introduced by piercing the fibers through the septa insert of the lids and making sure that the fibers were injected but not touching the media containing the bacteria. An internal standard was introduced 154 before sampling using 1 μL of the standard solution (10 mL/L of decane-d22 in ethanol) per jar. 155 For the anal sac samples, the swabs containing the anal sac fluid were placed in septa screw cap 156 jars that each contained a SPME. After a 24 hour incubation period, the SPMEs were removed. Then we 157 performed a liquid extraction of volatiles by adding 20 mL of methanol to the jars and incubating for 24 158 hours. 159 160 GC-MS analysis 161 Chromatography occurred on a 7890 GC (Agilent Technologies Inc., Santa Clara, CA) with a ZB-162 WAX 30 m × 250 μm capillary column, coated with a 0.25 µm film stationary phase (Part 7HG-G007-11, 163 100% polyethylene glycol from Phenomenex, Torrance, CA) equivalent to DB-Wax or Carbowax. Helium 164 was used as the carrier gas at 1 ml/min in constant flow mode. The inlet was set to 260 ℃ and SPMEs were 165 splitlessly desorbed during the run. The oven temperature was programmed to increase from 40 ℃ (held 166 for 5 min) to 110 °C at a rate of 5 °C min-1, and raise to 250 °C (held for 10 min) at a rate of 40 °C min−1.  The 16S rRNA gene PCR sequencing and analysis of the feline anal sac showed that 98% of the 191 reads that were placed into ASVs were assigned to six genera (Table 1). These ASVs generally represent 192 genera that contain anaerobic members known to be associated with mammals. Tessaracoccus species are 193 have been isolated in sediment and have also been found in the gut of mammals including rhinoceros and 194 humans [39][40][41][42][43]. Bacteroides is a genus of bacteria also often associated with mammals [44,45]. Anal sac secretion constituents and Bacteria isolates headspace SPME 237 We tentatively identified 127 compounds from the domestic cat anal gland secretion. Out of 127 238 tentatively identified compounds, 89 compounds were found in liquid extraction of anal secretion after 239 TBDMS derivatization (S1 Table), and 52 compounds were measured by SPME-GC-MS in the anal sac 240 secretion. These compounds were tentatively identified on the basis of the precise interpretation of its 241 accurate mass spectra, MS fragmentation, and Kovats index information. These VOC metabolites were 242 identified in the following compound chemical classes: heterocyclic compounds (12 %), alcohols (16 %), 243 fatty acids (17 %), ketones (11 %), aromatic carbons (13 %), amines (9 %), aldehydes (7 %), esters (6 %).

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A total of 67 compounds were tentatively identified from the SPME analysis of the three bacterial 245 isolates ( catus [20], A. melanoleuca [13], C. lupus [26], and M. furo [10]. Toluene is an aromatic compound found found in C. lupus [26]. Although anaerobic bacterial toluene degradation is a well known pathway [61], 258 toluene biosynthesis is less common in bacteria. It is possible that our tentatively identified result could be 259 a different compound but is likely to contain the alkyl benzene structure. Non-2-enal, and tetradecanal are 260 chain aldehyde compounds. Non-2-enal was previously found in C. lupus [26] and tetradecanal was found 261 in both S. suricatta [19,27], and A. melanoleuca [17] anal sac secretions. It is likely these aldehydes are 262 formed by bacterial oxidation of a ubiquitous fatty acid such as oleic acid [62]. Out of 52 compounds, six 263 fatty acids were tentatively identified from our anal sac secretion; nonanoic acid, pentadecanoic acid, trans-264 2-pentenoic acid, n-hexadecanoic acid, octadecanoic acid, and (Z)-docos-13-enoic acid. Fatty acids are one 265 of the most common compound groups known to be present in various animal anal sac secretions 266 [13,14,16,17,19,27] and all of the fatty acids found in our anal sac secretion have been previously reported 267 in other mammalian anal sac secretions. Nonanoic acid is a nine carbon fatty acid known to have unpleasant 268 rancid odor, and previously found in S. suricatta anal sac secretions [19]. Pentadecanoic acid was found in 269 P. leo [27], S. suricatta [19], and A. melanoleuca [13,17] [17] anal sac secretions. (Z)-docos-13-enoic acid was found in A. melanoleuca 272 [13,17] anal sac secretions. Fatty acids are a very common group biosynthesized by variety of organisms.

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The biogenesis typically starts with acetyl CoA, which is extended with malonate units to ultimately 274 assemble fatty acids containing an even number of carbons [62]. All six fatty acids found in our anal sac 275 secretion were also found in the Tessaracoccus sp UCD-MLA culture. It is possible that the highly abundant (in our sample) Tessaracoccus is largely contributing in the production of fatty acid compounds in this anal 277 sac. 278 41 compounds found in our bengal cat anal sac secretion have not been previously described in an 279 anal sac secretion. Cyclohexanone and dimethyl trisulfide were found from the anal sac secretion and also 280 from all three bacteria isolates. Cyclohexanone is known to be generated by the cyclohexanol degradation 281 metabolomic pathway, which is widely used by bacteria Aromatic compounds are common natural products in plants but also known to be produced by bacteria 294 [62]. Especially among the aromatic alcohols, such phenols, 2-penylethanol is one of the most aromatic 295 compound produced by diverse bacteria. In our present study, aromatic alcohols, such as phenyl methanol, 296 and 3,5-di-(tert)-buthylphenol were tentatively identified from both anal sac and bacterial isolates. These 297 aromatic alcohols can be assumed to be generated by the shikimate pathway which is present only in 298 microorganisms and plants, never in animals [67]. 299 300 301

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To our knowledge, this is the first study examining either the VOC profile of domestic feline anal 311 sacs or the VOC profiles of associated bacteria. We show that these particular feline anal sacs are dominated 312 by only a few taxa, most of which are easily culturable under anaerobic conditions. These bacteria produce 313 the majority of the identified volatiles in the total anal sac scent profile. Our preliminary identification of 314 these volatiles is supported by the existence of known bacterial metabolic pathways for the majority of 315 these compounds. Together these results support the fermentation hypothesis and suggest that further 316 characterization of the anal sac microbial community, as well as the VOC's produced therein could 317 potentially shed light on the potentially symbiotic relationship between these microbes and their host. 318 319 320 Acknowledgments 321 We thank Leah K. Isaacson, DVM for expressing the anal sacs and providing these samples to us.

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The authors would also like to thank Petra