Abstract
Felids urinate and spray ‘Marking Fluid’ for territorial maintenance and to transmit messages of their reproductive status. The very rare Himalayan snow leopard also utilises these two primary modes for chemical communication. The present paper is the first report on the volatiles in urine of snow leopards which were analysed with the help of headspace solid phase micro extraction gas chromatography mass spectrometry. Chemical profiles revealed the presence of numerous low molecular weight compounds with different functional groups like alcohols, aldehydes, ketones, sulphur containing compounds. Many monoterpene alcohols, which are common secondary metabolites of plants, are abundant in the urine collected during the months of October to December, the typical reproductive season of the snow leopard in the Darjeeling hills of the Eastern Himalaya. 6-Methyl-5-hepten-2-one was identified from this felid which has a characteristic odour perceptible by the human nose. Among many sulphur containing compounds, Dimethyl disulfide and Dimethyl trisulfide were common in all urine samples of both sexes. Saturated, monounsaturated and polyunsaturated fatty acids were also identified from the lipid fraction of the urine which, in nature, may play an important role by increasing the durability of the volatiles.
Access provided by Autonomous University of Puebla. Download conference paper PDF
Similar content being viewed by others
1 Introduction
‘Chemical signals’ which regulate a variety of physiological phenomena in many felids are the primary mode of information transfer related to the reproductive behaviour of these carnivores. (Albone 1984; Brahmachary and Dutta 1981, 1984; Wyatt 2014). All cat species, in general, have two modes of pheromonal communication, ordinary Urination and the spraying of Marking Fluid (MF) (Brahmachary and Dutta 1979, 1984, 1987; Brahmachary 1996; Brahmachary and Poddar-Sarkar 2015; Poddar-Sarkar and Brahmachary 2014). Alongside visual, auditory and tactile cues, members of the cat family predominantly use these two behavioural modes to mark their territory and to inform other individuals about their reproductive status. Thus, ‘scent marking’—differently termed as MF by us, plays a significant role in social interactions among snow leopards. Controversy regarding the origin of urine and MF in big cats existed for many years in international literature, however, it is now concluded that both these secretions are ejected through the urinary tract of these feline species (Poddar-Sarkar and Brahmachary 2014). In the present article, we try to identify those volatile and less/non-volatile chemical compounds in the urine of snow leopard, Panthera uncia syn. Uncia uncia (Schreber 1775) which may act as putative pheromones, although substantial evidence is yet to be required at this stage. Due to the extreme climatic conditions of the Darjeeling hills, unusual logistic constraints for work and strict zoo regulations hinder the authors’ intention for exhaustive work on this highly threatened animals. However, the authors intend to project their findings based on urinary volatiles which might be the first report on chemical communication of snow leopard in the Himalayas.
P. uncia is a crepuscular felid and the native to the North Eastern Himalayan mountain range of the Indian subcontinent (Fig. 1). Snow leopards generally lead a solitary lifestyle, rarely use audible sounds and exist in a very low population density in the Eastern Himalaya. In order to communicate with each other, snow leopards scrape the ground with their hind legs and spray urine against rocks to leave markings on the landscape (Sharma et al. 2006). Although the chemistry of urinary volatiles from other big cats such as lion, Panthera leo (Andersen and Vulpius 1999), tiger, Pathera tigris tigris (Poddar-Sarkar and Brahmachary 2014), bobcat, Lynx rufus (Mattina et al. 1991) and cheetah, Acinonyx jubatus (Poddar-Sarkar and Brahmachary 1997; Burger et al. 2006) were reported by many authors, little is known about the composition of snow leopard urine and the investigation of volatiles by headspace might be informative in the context of this species’ olfactory communication.
2 Materials and Methods
2.1 Collection of Samples
Urine was collected from snow leopards kept in open-air enclosures in the Padmaja Naidu Himalayan Zoological park (PNHZP), Darjeeling, West Bengal, India (27°03Wʹ30.1ʺN 88°15ʹ14.4ʺE). PNHZP is situated within the Darjeeling hill range (altitude 6,700 ft.) in the designated Eastern Himalaya Biodiversity Hotspot that has an undulated topography, an average temperature range during winter to summer of ~4–15.6 °C, a humid climate during the monsoon season with an average rainfall of ~309 cm and occasional snowfall during winter. After close observation of snow leopard behaviour, the schedule of urine collection was decided to be at dawn and dusk when the animals were fully active. Urine was collected from three female snow leopards (f1 = Studbook No: 2540, DOB 25.05.2004, f2 = Studbook No: 2399, DOB 29.03.2002, f3 = Studbook No: 2538, DOB 11.03.2004) on 17 occasions and from one male (m = Studbook No: 2404. DOB 08.07.2002) on 10 occasions following the procedure previously adopted by our team (Brahmachary and Dutta 1987; Poddar-Sarkar and Brahmachary 2014) over the years. Just before collection, animals were moved to a closed enclosure. Sampling schedule was rationalised for maximum collection opportunity and to maintain uniformity in experimental design covering both reproductive and non-reproductive seasons throughout the year during 2017–2018. Samples were pipetted out from the precleaned floor (only with distilled water) of the enclosure into 10 ml airtight Teflon-coated glass vials (Agilent, India), crimped immediately and transported to the laboratory under the ice. Samples for headspace volatiles (HSVs) were processed at the earliest convenience (i.e. between 72 and 96 h after collection) and samples for lipid work were kept at −20 °C for future analysis.
2.2 Chemical Analysis
Absorption of headspace volatiles (HSV) was optimised by attaching a 1 cm 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane [(DVB/CAR/PDMS); (Supelco, USA) stableflexTM, 24 Ga] SPME fibre with manual assembly holder (Supelco, USA) over the sample vials. Equilibration for absorbing the vapour phase was maintained at room temperature for 2 h in each case. Chemical analysis of HSV was performed using gas chromatography mass spectrometry [GCMS; Agilent 7890A, USA- triple axis MS-5975C] with a DB-WAX (30 m × 0.25 mm × 0.25 µm) column. Samples were desorbed for 10 min at the injector port at a temperature of 260 °C. The column temperature was maintained at 35 °C for 2 min initial hold, then ramping at a rate of 4 °C/min up to 210 °C with 3 min hold. The identity of the compounds was assigned by matching their retention time with authentic standards, whenever available [Sigma (USA) and by Dr. Ehrenstorfer GmbH, (Germany)] (Table 1) as well as by co-chromatography and by comparison of their respective mass spectral data obtained from the NIST (2011) library, by calculating Linear Retention Index (LRI) in relation to n-alkanes of C11–C19, considering the EI-MS fragmentation pattern and from previous records of this laboratory. The flow rate of the carrier gas (Helium) was maintained at 1 ml/min. The front inlet temperature was 250 °C. The temperature of the MS source, quadrupole and auxiliary heater were set at 230 °C, 150 °C and 280 °C, respectively. The electron energy was 70 eV (vacuum pressure-2.21 e-0.5 torr). The mass fragment scan range was 50–450 amu at 0.5 s/scan.
Lipids were extracted from the urine by using the Bligh and Dyer’s (1959) method. An aliquot of chloroform extract was taken in a pre-weighed glass vial for gravimetric estimation of lipids. The solvent was evaporated to dryness with a stream of N2 and the residue weighed again by precision balance. For the analysis of fatty acids (FA), the chloroform phase was used. The fatty acids present in urine were derivatized to fatty acid methyl ester (FAME) by acid catalysed esterification (Poddar-Sarkar 1996). FAME was recovered with n-hexane and finally dried over anhydrous sodium sulphate. The volume of the n-hexane was reduced under a stream of nitrogen and subjected to GCMS. 1 μl of hexane extract of FAME was injected to HP5-MS column (30 m × 0.25 mm × 0.25 μm) of GCMS (Agilent Technologies, USA; 7890A GC system with 5975C triple axis detector MS) apparatus. The programme was set at 70 °C initial hold for 1 min for column temperature, ramping at 4 °C/min. up to 260 °C with a final hold for 3 min. FAME were identified by calculating their relative retention time (RRt) and comparison with authentic mixture of 37 FAME and PUFA (Supelco, Lot No: LB80556 and LB77207, USA). Identification was confirmed by comparing mass fragmentation pattern of the compounds from the NIST (2011) data base.
2.3 Statistical Data Analysis
A total number of 27 samples from three females and one male were analysed by GCMS. For quantitative analysis, each peak was normalised by calculating the relative percentage considering total ion count from the chromatogram. Chemical compounds identified by mass fragmentation pattern were grouped into different classes on the basis of their functional group or nature of backbone. Summation of nine classes of compounds from all females during the reproductive season (RS) and non-reproductive season (NRS) were plotted in Fig. 2. In addition, a comparative assessment on the basis of such 35 identified volatile compounds which were present in all urine samples of four leopards were done by heat map (Fig. 3). For heat map generation, successive steps were followed: Step (i) the sampling events were segmented into two seasons: RS and NRS for each animal; (ii) A table was developed by considering average amount for each identified compounds taking all females in a pool and for data in male in separate pool; (iii) Values were converted to the percentage of the row sum of each compound considering NRS & RS separately for female pool as well as for male pool; (iv) Derived values were used to form the final matrix (Fig. 3). Statistical data were processed using past software (3.21 version) for generating the heat map.
3 Results
A number of volatile organic compounds (VOCs) with different functional groups like alcohols, aldehydes and ketones were identified from the HSVs of urine of snow leopard of both the sexes (Table 1) The most interesting compound 6-Methyl-5-hepten-2-one which has a characteristic aroma, perceptible by the human nose was emitted from the fresh urine of snow leopard. However, no such distinctive aroma was perceptible from the distilled water washings of the floor processed in the same manner as a control, and 2-acetyl-1-pyrroline, the aroma molecule responsible for the characteristic smell of ‘Basmati rice’ present in MF of tigers and Indian leopards (Brahmachary et al. 1990; Brahmachary 1996; Poddar-Sarkar and Brahmachary 2014) was not detected in the urine of snow leopards . Dimethyl disulphide and Dimethyl trisulfide , two sulphur compounds were also identified from urine of snow leopard (Table 1). Low boiling straight chain alcohol of carbon number 5, 6, 7, 8 and aldehyde of 6, 8, 9 were common HSVs present in the urine of both male and females. Two carboxylic acids, such as acetic acid and 4-hydroxy butanoic acid were identified from both sexes. Some urinary constituents like phenol, benzaldehyde, p-cresol, acetophenone were also identified from urine. Some compounds which are very common secondary metabolites of plants like azulene, 1-methyl-2-piperidone, beta-ocimene, p-cymene, p cymenene were also identified in urine of snow leopard. HSV profile of the urine collected from reproductive season showed significant presence of some terpenoids such as alpha-terpineol, gamma-terpineol, terpineol-4-ol, cis-dihydro-alpha-terpineol and terpinolene (Table 1). We found distinctive variations in the relative abundance of some compounds in both sexes during RS and NRS. During RS, high amounts of monoterpene alcohols and aromatic alcohols were identified in contrast to the lower amounts of sulphur and nitrogen containing compounds as well as aliphatic alcohols (Fig. 2). RS and NRS differed between male and female urine (Fig. 3).
Lipids present in the urine of snow leopard of both sexes ranged from 0.95 to 1.42 mg/ml. The Lipid fraction of snow leopard urine mostly contained Saturated Fatty Acids (SFA) of even and odd carbon number. Palmitic acid (16:0) is the most abundant in all cases (Fig. 4). In addition to even carbon number SFA such as Decanoic acid (10:0), Dodecanoic acid (12:0), Tetradecanoic acid (14:0), Octadecanoic acid (18:0), Eicosanoic acid (20:0), Docosanoic acid (22:0) and Tetracosanoic acid (24:0) some SFA with odd carbon number, such as Tridecanoic acid (13:0), Pentadecanoic acid (15:0), Heptadecanoic acid (17:0), Nonadecanoic acid (19:0) and Heneicosanoic acid (21:0) were also identified. In addition, Benzeneacetic acid is also detected in the urine of snow leopard. Five monounsaturated FAs (7-Hexadecenoic acid, 9-Hexadecenoic acid, 9-Octadecenoic acid,11-Eicosenoic acid and 13-Docosenoic acid) and two polyunsaturated FAs (9,12-Octadecadienoic acid and 5,8,11-Eicosatrienoic acid) were identified (Fig. 4).
4 Discussion
One of the volatiles of fresh snow leopard urine is sulcatone (6-Methyl-5-hepten-2-one), which imparts a characteristic odour perceptible to the human nose. Urine of snow leopards contains many characteristic low molecular weight compounds with diverse functional groups such as pentanol, hexanol, heptanol, 3-octanone, nonanal, indole, etc. which might play a role in chemical communication. Similar types of compounds have been shown to moderate and govern a variety of specialised behaviours related to kin recognition, choosing potential partners and maintaining social standings in many mammals including other felids and canids (Andersen and Vulpius 1999; Burger et al. 2006; Raymer et al. 1984; Soso and Koziel 2017; Wilson 1980). Dimethyl disulphide (DMDS), a male attractant compound of Hamster Vaginal Secretions (Singer et al. 1976) and one of the most important constituents of MF in lions (Soso and Koziel 2017) as well as Cheetah urine (Burger et al. 2006) was also confirmed in the urine of Snow leopard. Many secondary metabolites of plant systems were identified from the urine of snow leopard, and we observed fragmented leaves in their scats. Interestingly, we found significant variation in urinary terpenoidal compounds during their reproductive season. Hexanal, Octanal, Nonanal, 4-heptanone and benzaldehyde, identified from urine of both female and male snow leopard were considered as common urinary volatiles of many mammals such as the house mouse (Novotny et al. 1999), white-tailed deer (Miller et al. 1998) and elephant (Rasmussen and Greenwood 2003), coyote (Schultz et al. 1988), ferret (Zhang et al. 2005) and MF of lions (Soso and Koziel 2017). Other low carbon alcohols and aldehydes are also common urinary volatiles of many mammals (Albone 1984). Phenethyl alcohol, detected in urine of snow leopard, is one of the major volatiles of lion MF (Soso and Koziel 2017). Fatty acids, identified from the urine of snow leopard are similar in nature to other big cats such as tiger, leopard, lion and cheetah (Poddar-Sarkar 1996; Poddar-Sarkar and Brahmachary 2014). It can be assumed that lipids may be delaying the dissipation of urinary volatile constituents which may facilitate animals to mark vast areas for territorial maintenance (Brahmachary and Dutta 1987; Poddar-Sarkar and Brahmachary 1996; Poddar-Sarkar 1996; Poddar-Sarkar and Brahmachary 2004). Brahmachary and Dutta observed previously that steam distillation separates the smell of volatiles which rapidly vanishes after being liberated from the heavier lipids (Brahmachary unpublished; Brahmachary and Choudhuri unpublished; Brahmachary and Dutta 1979; Poddar-Sarkar and Brahmachary 2014). As urine is one of the major sources of pheromone in other felids, it can be presumed that it might play a similar role in snow leopards. Nevertheless, extension of this work may add some new findings in the future. Therefore, by analysing the VOCs of urine throughout the year, the physiological status of the animal can be assessed and could form an important basis for the planning and management of future breeding programmes of this rare species as well as being utilised for zoo management and conservation purposes.
References
Albone E (1984) Mammalian semiochemicals. Wiley, Chichester, UK
Andersen KF, Vulpius T (1999) Urinary volatile constituents of the lion. Chem Senses 24:179–189
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Brahmachary RL (1996) The expanding world of 2-acetyl-1-pyrroline. Curr Sci 71:257–258
Brahmachary RL, Dutta J (1979) Phenylethylamine as a biochemical marker of tiger. Zeitschrift für Naturforschung C 34:632–633
Brahmachary RL, Dutta J (1981) On the pheromones of tigers: experiments and theory. Am Nat 118:561–567
Brahmachary RL, Dutta J (1984) Pheromones of leopards: facts and theory. Tiger Paper 11:18–23
Brahmachary RL, Dutta J (1987) Chemical communication in the tiger and leopard. In: Tilson RL, Seal US (eds) Tigers of the world: the biology, biopolitics, management, and conservation of an endangered species. Noyes Publications, Park Ridge, NJ
Brahmachary RL, Poddar-Sarkar M (2015) Fifty years of tiger pheromone research. Curr Sci 108:2178–2185
Brahmachary RL, Poddar-Sarkar M, Dutta J (1990) The aroma of rice … and tiger. Nature 344:26
Burger BV, Visser R, Moses A, Le Roux M (2006) Elemental sulfur identified in urine of cheetah, Acinonyx jubatus. J Chem Ecol 32:1347–1352
Mattina MJI, Pignatello JJ, Swihart RK (1991) Identification of volatile components of bobcat (Lynx rufus) urine. J Chem Ecol 17(2):451–462
Miller LA, Johns BE, Elias DJ (1998) Immunocontraception as a wildlife management tool: some perspectives. Wildl Soc Bull 26:237–243
Novotny MV, Ma W, Wiesler D, Zidek L (1999) Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein. Proc R Soc Lond B 266:2017–2022
Poddar-Sarkar M (1996) The fixative lipid of tiger pheromone. J Lipid Mediat Cell Signal 15:89–101
Poddar-Sarkar M, Brahmachary RL (1997) Putative semiochemicals in the African cheetah. J Lipid Mediat Cell Signal 15:285–287
Poddar-Sarkar M, Brahmachary RL (2004) Putative chemical signals of leopard. Anim Biol 54(3):255–259
Poddar-Sarkar M, Brahmachary RL (2014) pheromones of tiger and other big cats. In: Mucignat-Caretta C (ed) Neurobiology of chemical communication. CRC Press/Taylor & Francis, Italy
Rasmussen LEL, Greenwood DR (2003) Frontalin: a chemical message of musth in Asian elephant (Elephas maximus). Chem Senses 28(5):433–446
Raymer J, Wiesler D, Novotny M, Asa C, Seal US, Mech LD (1984) Volatile constituents of wolf (Canis lupus) urine as related to gender and season. Experientia 40(7):707–709
Schultz TH, Flath RA, Stern DJ, Richard MT, Teranishi R, Kruse SK, Butler B, Howard WE (1988) Coyote estrous urine volatiles. J Chem Ecol 14:701–712
Sharma S, Dutta T, Veer bhatnagar Y (2006) Marking site selection by free ranging snow leopard (Uncia uncia). In: McNeely JA, McCarthy TM, Smith A, Olsvig-Whittaker L, Wikramanayake ED (eds) Conservation biology in Asia. Nepal: Society for Conservation Biology Asia Section and Resources Himalaya
Singer AG, Agosta WC, O’Connell RJ, Pfaffmann C, Bowen DV, Field FH (1976) Dimethyl disulfide: an attractant pheromone in hamster vaginal secretion. Science 191(4230):948–950
Soso SB, Koziel JA (2017) Characterizing the scent and chemical composition of Panthera leo marking fluid using solid-phase microextraction and multidimensional gas chromatography–mass spectrometry-olfactometry. Sci Rep 7(1):513
Wilson EO (1980) Caste and division of labor in leaf-cutter ants (hymenoptera: formicidae: Atta): I. The overall pattern in A. sexdens. Behav Ecol Sociobiol 7:143–156
Wyatt TD (2014) Pheromones and animal behavior: chemical signals and signatures, 2nd edn. Cambridge University Press, Cambridge, UK
Zhang JX, Soini HA, Bruce KE, Wiesler D, Woodley SK, Baum MJ, Novotny MV (2005) Putative chemosignals of the ferret (Mustela furo) associated with individual and gender recognition. Chem Senses 30(9):727–737
Acknowledgements
Author SD [CSIR sanction no-09/0289(0996)/2017-EMR-1], SM [09/0289(1004)/2017-EMR-1] and PD [108(Sanc.)/ST/P/S&T/1G-24/2014] are grateful to Council of Scientific & Industrial Research (CSIR), Government of India and Govt. of West Bengal respectively for providing their fellowships during this work. We would also like to acknowledge the Department of Science and Technology (Fund for Infrastructure development in Science and Technology programme) Govt. of India for extending GCMS facility in the Department of Botany, University of Calcutta. We also acknowledge the kind help and assistance from Principal Chief Conservator of Forest (wildlife), Govt. of West Bengal, and Director of Padmaja Naidu Himalayan Zoological Park, Darjeeling, West Bengal.
Note:
We dedicate this paper to the memory of our mentor Late Prof. R.L. Brahmachary with our deep grief and sorrow. He corrected our initial draft of this manuscript but passed away on 13 February 2018 when we were submitting the final version of this paper.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Nature Switzerland AG
About this paper
Cite this paper
Das, S. et al. (2019). Do Urinary Volatiles Carry Communicative Messages in Himalayan Snow Leopards [Panthera uncia, (Schreber, 1775)]?. In: Buesching, C. (eds) Chemical Signals in Vertebrates 14. Springer, Cham. https://doi.org/10.1007/978-3-030-17616-7_3
Download citation
DOI: https://doi.org/10.1007/978-3-030-17616-7_3
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-030-17615-0
Online ISBN: 978-3-030-17616-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)