Scientists in bulk inhabit a city of water tight compartments ... but
by the irony of the situation, the compartments are not quite water
tight...
Julian Huxley
This is the true story of a research that led me from the tiger to basmati rice to mung bean and the tiger, too, stepped in, being helped by a snail.
My story started on a hot summer day in (now famous) Kanha, India, in 1964. George Schaller (who had just commenced the first long term scientific research on the tiger) and I noticed an Indian file of spotted deer behaving in a curious manner. Each one stopped in front of a small Butea monosperma shrub and sniffed and sniffed, then followed by the next in the line and so on. We then investigated and noticed that the leaves of the shrub were heavily charged with "tiger smell". Much later I realised that that was a billet doux of a love-lorn tigress who had been dreaming of Prince Charming in her mid-summer night's dream.
Only a few days ago, Schaller saw a tigress pausing from time to time along her way and jetting a stream of fluid against a rock or tree. Now, in more than a century of blood-sport literature this was never mentioned. Stalwarts of a bygone era, Baker, Inglis, Brander or Corbett apparently knew nothing of this fact. In 1954 Locke in Malay first recorded this tiger spray but it was Schaller who succeeded in bringing it to the notice of scientists in 1967. Locke surmised the source of the spray to be a gland at the tail-root and Schaller thought the fluid was a mixture of urine and anal gland secretion. Both were wrong.
In those days Schaller was toying with the idea that tigers might leave messages through this odorous fluid. I picked up a Butea leaf and for the next ten days the tiger-smell on it was still perceptible to me.
It is well know that smelly signals (termed pheromones) play an important role in the world of insects. The male emperor moth may perceive bombycol, the pheromone of the female, from a distance of 2km or so. But in 1964 our knowledge of mammalian pheromones was scanty. Two of these, muscone from the male musk deer and civetone from the civet, had been chemically studied. Muscone was supposed to attract the female musk deer. Masefield's poem describes the olfactory signal of the vixen:
A faint rank taint like April coming
It cocked his ears and his blood went drumming
For somewhere out in Ghost Heath Stubbs
was a roving vixen wanting cubs.
Over the valley floating faint
on a warmth of windflow came the taint.
Again, everybody has seen a dog spraying urine against a tree or lamp-post, with a hind leg raised. This was believed to be a signature, a notice-board or placard staking a claim to his territory. Many have also noticed the tom cat spraying urine. So, Schaller's suggestion that tigers and tigresses might advertise their presence (territorial claim and/or nuptial invitation) in this manner seemed reasonable to me. (Later I leant that of all the big cats only in the tiger the female of the species takes strong initiatives in spraying.) The tigress is more of a hussy, blatantly drawing attention to herself by spraying while the lioness or leopardess is more demure in this respect. Schaller's view was all the more imprinted in my mind because I was working on "chemical signals" in the pond snail embryo and that time (and later on). Such signals are transmitted from the nucleus to the cell cytoplasm (messenger RNA) and signals from cell to cell to keep them aware of each other. One step removed from such cell-to-cell message is the chemical communication from an ant to ant. Precisely then (1964 and onwards) Wilson chemically identified such signals in the ant. Another step and we have pheromonal messages transmitted over longer distance in the world of tigers. The microscopic embryo cells in my laboratory and the tiger burning bright in the forest of the night seemed to be united by a common thread. But I had to wait for a dozen years before I could develop the idea any further.
Years later Khairi, a tigress, was raised by S R Chaudhuri and still later Tara, another tigress, was reared by Arjan Singh. (This latter was a more satisfactory case because Tara's habitat was all along the natural forest.) In May, 1976, I met Khairi, a nearly full-grown tigress, during her brief sojourn in the heart of the Indian jungle. Once again I picked up a Butea leaf charged with her spray-fluid and marvelled at the beautiful fragrance of this very fresh deposit. On that May-day I chalked out a programme for working on the smelly signals of tigers as barbets ceased their calls and peafowl began miaowing on roosting trees.
Fragrance of flowers, stinking sewers, roasting steak, fried spices like cumin or coriander - we perceive a host of odour nuances. What is a smell? The relatively small molecules of certain substances (molecular weight not exceeding 300) fly through the air, bind to large "receptor" molecules (at least of !000 types) of our nasal epithelium - and hey presto, we smell. As surprisingly small amounts of odorous substances (sometimes a few or even a single molecule) generate the sense of smell it may be very difficult to identify their chemical nature. In the last few decades this study has been facilitated by the use of certain apparatus but even now human nose is superior to any device designed by human ingenuity. But human nose is so very blunt compared to that of many animals!
Jim Corbett, the great hunter-naturalist, repeatedly and unequivocally stated that tigers have no sense of smell. This is patently wrong. Even if the tiger does not use its olfactory power while hunting - and this itself has been questioned by several knowledgeable old-timers - smell certainly plays a role in other aspects of his life. Those who have observed pet tigers from close quarters, such as Chaudhuri, Arjan Singh, Lindblad, Tippy Hedren (of Hitchcock film fame) and, to a lesser extent, myself - all of us noticed a certain degree of olfactory ability in the tiger. Today we also know that when a tiger/tigress perceives a smell he/she executes a "grimace" with the tongue lolling. This gesture, known as flehmen, forces some of the smell molecules into the vomeronasal organ (VNO). Then the animal tests the source of smell with the tongue and the tongue-tip transfers some more molecules (which do not fly through the air) into the pair of long, thin VNO tubes. VNO has 200 types of receptor molecules. This aspect of flehmen has been understood since 1972.
Thanks to the pet tigress we gained a clear concept of the spray. Urine drips downwards and the animal, while urinating, lowers the hindquarters but this marking fluid (MF) is sprayed upwards and backwards while the tiger or tigress remains standing. It is the same for the lion. The leopard and cheetah sit on their haunches while urinating but they also stand when spraying. The MF is ejected through the urinary tract and there is no channel connecting it with the anal-glands as determined by Hashimoto and others. The secretion of the anal gland might find its way into faeces but not urine. In 1997 I examined the MF and anal gland secretion of an Asiatic lion. Chemically, they seem to have some differences. There is of course no gland at the root of the big cats' tails.
When ten tigers and tigresses at Nandan Kanan Zoological Park, Orissa, India, sprayed a total of 9662 times, they tallied only 189 urinations. This alone suggests that MF must have evolved to some purpose, otherwise such wastage would have been selected against. I have also noticed a similar preponderance of MF spray in the Asiatic lion, Indian leopard and African cheetah but the females of these species rarely jet. I collected thousands of data and Nandan Kanan which suggest that MF spray is likely to be meant for other tigers to decode.
Together with my colleagues, most J Datta and (Ms) Mousumi Poddar-Sarkar, I have been busy analysing and identifying the chemicals which are used as signals. All volatile molecules - and all of these have some odour or other - are putative candidates. So, our first step was to separate the volatile molecules with the help of steam distillation. Straightforward, though laborious, methods pursued over the years revealed the identity of many molecules. We found a dozen fatty acids, one of which, acetic acid, is known to the layman as vinegar in the kitchen. Then there are many amines, also aldehydes, and we found acetone. But the most challenging and, therefore, the most rewarding was a slippery eel of a molecule that eluded us for 19 years. This is 2 acetyl-1-pyrroline (2AP), the very same molecule which imparts the beautiful aroma to fragrant varieties of rice (such as basmati). When we first started our work this molecule was unknown. As late as in 1982, American scientists headed by Buttery discovered 2AP and the next year they traced it to Pandanus foetoedus leaves. In some parts of Asia these leaves used to be boiled with ordinary rice so as to impart a good aroma. In the 1970's I did some preliminary experiments which suggested certain chemical affinities between P. foetoedus aroma and tiger MF aroma. (It occurs also in the ordinary urine of the tiger.)
At last we established the presence of the identical molecule in fragrant rice and tiger MF. This was the first know example of 2AP occurring in the animal kingdom, incidentally, in our National Animal. (Put a tiger in your kitchen - vinegar and basmati aroma assured!)
In 1997, S Midya and I detected this molecule in the night-blooming flower of Bassia latifolia (= Maduca indica/longifolia). This is the first example of 2AP occurring in the floral kingdom. From 1997 to 1999 Mahua Ghosh and I have been busy with the aroma of Mung bean. In Bengal there was a strain of mung bean endowed with an unusually beautiful aroma (apparently very different from rice fragrance) but this strain is practically extinct now. We collected several strains; the two best qualities possess, among other compounds, the very same 2AP. The other compounds alter the tone or quality of the aroma. Most of these occur in ordinary strains of mung bean also. We have identified about half a dozen of these, two of which are pheromones in rodents (a male-attractant vaginal pheromone and a puberty delaying pheromone). The occurrence of these pheromone molecules in boiled or fried mung bean is coincidental but some varieties of our local rice develop the smell while ripening or even before that. I do not know what the utility of this aroma in the rice plant or Bassia flower is, but I have a hunch - this might be the arsenals to fight against some fungi or bacteria. Such is the case in the Jasmine, Bassia flower is bactericidal and strong rust fungus resistance is known in fragrant rice in Bengal.
Schieberle synthesised 2AP in a relatively simple way, by heating proline, an amino acid (more pedantically, imino acid) with sugar (glucose or fructose or sucrose) at a temperature of 170°C. Over two years my colleagues (Mostly Ms Nila De Sarkar) and I altered the conditions and finally could bring the temperature down to 105°C but no lower. We do not know by what mechanism the tiger or the rice plant or Bassia flower biosynthesise the aroma at a much lower temperature (in autumn sunshine and spring night, respectively). Again, some molecules occur in two forms like left handed and right handed gloves or shoes. In the case of at least two compounds, Carvone and Limonene, the quality of aroma depends on this "handedness". I synthesised 2AP from these two types of proline. Here, the quality of smell does not depend on handedness. (With decreasing concentration of 2AP we perceive, however, significant changes in the tone, from extreme sweetness to a different, rice-like fragrance.)
Apart from the tiger, I have also studied the Asiatic lion, the Indian leopard and African Cheetah. Tiger and leopard spray upwards and backwards, the cheetah switches the spray up and down and the Asiatic lion is very variable in this respect - spraying upwards and backwards, switching up and down, downwards vertically, downwards at an angle and horizontally backwards. The fatty acid pictures of the tiger, leopard and cheetah MF are similar. 2AP occurs in the leopard but not in the lion (Asiatic or African) or cheetah. Four tiger cubs (one male and three female) executed the first flehmen gesture in the fourth month. Presumably, then the olfactory ability started developing. Two cubs described by Corbett (Temple Tiger) may have failed to detect the smell of rotting meat because they were below that developmental stage. Adult tigers and African lions I observed at Adamson's camp in Kora, Kenya, perceive high game or rotting meat, but generally not fresh meat, by olfaction. During my second visit to Adamson's camp (1988-89) I observed three lion cubs during their fifth and sixth month of life. In the second half of the fifth month they suddenly became conscious of smell and frequently flehmened after sniffing urine etc. They also precisely then began rolling on the droppings of dik dik, the smallest antelope of that region. Adult lions do so.
In a male tiger cub (Dora 3) I had with me at Nandan Kanan the first weak attempt of spraying was noticed at seven months of age and in the African lion cubs, in the fifth month. The first real spray by Dora 3 was seen at about one year of age and 2AP appeared in the urine and MF at this time. In the leopard Cub (Chitro) 2AP appeared in the urine at the age of 3-4 months but there was no aroma at the age of one month.
Perfumes we use have been designed (by perfumers) to linger on for some time. Without a sticky substance to delay the rate of the volatile molecules flying away, the smell would rapidly disperse. The tiger or, Nature, has devised such a "gum", namely lipid (fat) molecules. M Poddar-Sarkar has worked out the nature of these molecules.
Schaller noticed that the tiger smell would last for days and weeks unless a very strong shower washes it off. I kept hanging under the tap, overnight, a clump of such smelly leaves. The next day the smell was still strong on the lower leaves. I kept a clump of such leaves (only some of which were smelly) immersed in estuarine water in a creek for 22 hours and after that two persons (unbiased) immediately and unerringly pointed out which particular leaves had been charged with the smelly fluid. The "lipid gum" had fixed the volatile molecules for 22 hours.
Over hours and over days as the signalling molecules escape it may become more and more difficult to read the message. Furthermore, many animals "overmark", ie another tiger, say, while passing by sprays over the resident's marking; the resident, on coming back, is likely to overmark his overmarking. Among some rodents it has been shown that a third animal can distinguish between the odour of the two animals in the over-marking and it evinces a preference for the top marking! How the distinction is made even when the overmarking totally overlaps the earlier marking is difficult to imagine. It seems to be a well-nigh incredible feat on the part of the animal.
How can the tiger distinguish between the smell of two or more neighbours' MF? That the police dog can search out the criminal from a population of hundreds of men proves that, like fingerprint, body smell is also unique, characteristic for every individual. But the odour of sweat, say, is composed of the same fatty acids in all of us. But perhaps the ratios and proportions vary. Forman found such characteristic proportions in each member of a group of 24 mongoose. Recently, similar results have been reported in more animal species. Our results, with three tigers, a mother and son duo and a third distantly related tigress, are more complicated. Over a prolonged time period the ratios vary even in the same animal. Nonetheless, the profiles of the mother and son are closer than with respect to the third. So here, too, we have an inkling of the possible mechanism.
I have gone far since that summer day of 1964, or that Mayday of 1976. Watching in Nature led to close observations on captive animals (which should be rehabilitated in their real habitat, the jungle and the bush as Adamson and Arjan Singh tried to do) and then to laboratory work and theoretical thought processes. From snail to tiger to mung bean is apparently a tortuous path but now, in retrospect, it seems to have been a smooth, natural course that reached up to the end of the century.
I had to face stiff resistance in the course of these researches. All this was man-made and turned out to be more forbidding than the difficulty intrinsic in my complex research problem. Of those who supported me:
I must first mention Prof. P. C. Mahalanobis whose farsighted and visionary approach enabled me to work at all on embryology and then on tiger pheromone in the Indian Statistical Institute. This tiger research would not have been possible but for the co-operative attituide of the late S. R. Choudhuri and S. K. Patnaik, the then director of Nandan Kanan. I am grateful to the late Mrs Indira Ghandhi who took prompt steps to ensure adequate funding at one stage of the research; to Nihar Nalini, George Schaller, George Adamson, Tony Fitzjohn, Gareth Patterson, Doddie, Julie, several workers at Kora, Africat of Namibia, Von der Becke and several persons in Virunga, Zaire, Some staff members of Nandan Kanan, some workers of ISI, A. Sreevastav, M. Singh, P. Amte, WWF Calcutta, Dr. Sabapura, Manisha Rajput, Sarada Mallia, Sally Walker, USIC of Calcutta University, several scientists and research scholars of Applied Chemistry (Calcutta University), Prof. P. Bhargav, Dr. Schieberle (Germany), Drs. Sommerville, Waterhouse, Francis (Cambridge), scientists at IICB at Jadavpur (especially Dr. E. Ali and Dr. P. Bhattacharya), several scientists and technicians of Bose Institute Calcutta, Susmita, Anuradha, Ketaki, Sekhar, Manikanchan and some others. With the help of all these people I have been able to defeat a nexus of vicious people and bring the 25-odd-year long quest to a reasonably satisfactory end.
Marking fluid could very easily be collected from Khairi and a tame cheetah at Namibia and the tame leopards of P. Amte. Over the years we perfected a method of collecting MF from tigers at Nandan Kanan which was also successful in the case of Asiatics lions in Gir, India and Indian leopard at Buxa, North Bengal. This method is best explained through a video and some photographs.
Most of the techniques we have used for chemical analysis are paper chromatography, thin layer chromatography and gas chromatography. We have been lucky to find GCMS (gas chromatography and mass spectroscopy) and HPLC (high pressure liquid chromatography) facilties in Calcutta and Bangalore. These are difficult to get in the third world. At Cambridge sniff GLC & GCMS were available.
We published 20 papers on the topic described here; the 6 most important are in American Naturalist 118, 561 (1981); Endeavour, New Series 10(2) 1986; Chemical Signals in Vertebrates VI (1992) & VII(1995); Nature 344, 26 (1990); Current Science, 71, 256 (1996).
Bibliography of Prof. R. L. Brahmachary's Papers, compiled by David R. Kelly
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