Bacterial Action and Chemical Signalling in the Red Fox (Vulpes

5 Bacterial Action and Chemical Signalling in the Red

F o x (Vulpes vulpes) and Other Mammals

ERIC S. ALBONE—Department of Animal Husbandry, University of Bristol, Langford Bristol BS18 7DU,

U.K.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2017 | http://pubs.acs.org Publication Date: February 2, 1978 | doi: 10.1021/bk-1978-0067.ch005

PAULINE E . GOSDEN and GEORGES C. WARE—Department of Bacteriology, University of Bristol, Bristol BS8 1TD, U.K. DAVID W. MACDONALD and NICHOLAS G. HOUGH—Animal Behavior Research Group, Department of Zoology, University of Oxford, Oxford OX1 3PS, U.K.

The products of microbial action play an important part i n food attractancy. Man has long employed micro-organisms to contribute characteristic flavor q u a l i t i e s to foodstuffs (1,2,3). For various carnivores also, odors substantially of microbial o r i g i n can be attractive. The attractancy exhibited by the scent of decomposing carrion may be related to the use of odor cues to locate food in the natural environment. However, the phenomenon i s complex. Inedibly rotten meat constitutes an effective lure to trap fox and, although edible carrion can provide an important food source for the red fox, highly putrefied carcasses possessing great attractancy are not consumed but instead frequently become important scent marking sites (4). In addition to such "environmental" scent sources, microbial a c t i v i t y i s also associated with the body surfaces of l i v i n g mammals themselves. Recently it has been suggested that microbially generated odors arising from such sources could serve a communicative function i n certain species. This concept has been discussed by Alboneetal,1977 (5). Although i n the context of mammalian chemical communication generally, v o l a t i l e substances have received major attention, i n v o l a t i l e materials could also be important i n cases where physical contact occurs(6). Examples of such contact are many and include the l i c k i n g responses to vaginal secretion i n the golden hamster (7), to urine i n the red fox (4), and to the t a r s a l scent organ i n the blackt a i l e d deer (8). Further, i n v o l a t i l e components of guinea pig urine have been implicated i n sexual recognition (9), while the puberty-accelerating pheromone i n male mouse urine i s also thought to lack v o l a t i l i t y (10) . The interdisciplinary study of communication by chemical signals in mammals is itself new (11, 12), while an exploration of the role of bacteria i n generating such signals i s only now commencing. In this paper, we offer a necessarily incomplete discussion based on the results of experiments, undertaken i n a variety of disciplinary contexts, on microbially generated chemical signals of environmental and mammalian o r i g i n , with ©

0-8412-0404-7/78/47-067-078$05.00/0

Bullard; Flavor Chemistry of Animal Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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p a r t i c u l a r reference t o the case o f red f o x . Such s t u d i e s , w h i l e being o f fundamental importance t o a proper understanding o f t h e b i o l o g i c a l f u n c t i o n o f o l f a c t o r y communication, are a l s o o f p o t e n t i a l a p p l i e d importance f o r predator c o n t r o l and p u b l i c h e a l t h programs. An example o f the l a t t e r would be i n the development o f e f f e c t i v e b a i t s f o r the o r a l a d m i n i s t r a t i o n o f r a b i e s vaccine t o w i l d fox populations as p a r t o f an a n t i - r a b i e s program (13, 61).


Fermentative Scent Sources: I) Mammalian;

the A n a l Sac

In mammals, m i c r o b i a l odor production i s most l i k e l y t o r e s u l t from incomplete substrate o x i d a t i o n a s s o c i a t e d w i t h anaerobic processes, the end products o f aerobic catabolism being p r i n c i p a l l y carbon d i o x i d e and water. As a r e s u l t , e x t e r n a l anatomical s t r u c t u r e s such as pouches or c a v i t i e s , p a r t i c u l a r l y i f f l u i d f i l l e d , are o f major importance as these hinder access o f a i r and maintain warm, moist (or wet) c o n d i t i o n s f a v o r i n g m i c r o b i a l growth. The anatomical l i t e r a t u r e r e v e a l s numerous such s i t e s , although very few have yet been s t u d i e d i n t h i s context. Examples i n c l u d e the i n f r a - o r b i t a l and i n t e r d i g i t a l glands o f ungulates, the p r e p u t i a l d i v e r t i c u l u m o f the p i g , the anal sacs o f c a r n i vores and the perfume pockets o f v i v e r r i d s (14,15) . The vagina i s another important fermentative scent source which has been s t u d i e d i n r e l a t i o n t o chemical s i g n a l l i n g i n p r i m a t e s , p r i n c i p a l l y by Michael and h i s a s s o c i a t e s (16). I t i s i n t e r e s t i n g t h a t although very few compounds have yet been i d e n t i f i e d t o which a s p e c i f i c mammalian communicatory f u n c t i o n can be assigned, a number o f these are substances commonly occur i n nature as m i c r o b i a l products. Among these are dimethyl d i s u l f i d e , a sex a t t r a c t a n t i n hamster v a g i n a l s e c r e t i o n (17) and a common product o f the m i c r o b i a l degradation o f organic matter (18,19) and p h e n y l a c e t i c a c i d and i s o - v a l e r i c a c i d , common fermentation productions o f amino-acids (20) which have been i d e n t i f i e d a l s o i n the v e n t r a l gland o f the Mongolian g e r b i l (21) and i n the s u b a u r i c u l a r gland o f the pronghorn (22) r e s p e c t i v e l y . In none o f these cases does the p o s s i b i l i t y o f a m i c r o b i a l o r i g i n appear t o have been i n v e s t i g a t e d , however. The anal sac c o n s t i t u t e s an important fermentative scent source i n the c a r n i v o r e s . I n the red f o x , the two anal sacs form r e s e r v o i r s o f about 1 ml c a p a c i t y s i t u a t e d l a t e r a l l y t o t h e anus between the i n t e r n a l and the e x t e r n a l anal s p h i n c t e r muscles. Each sac opens t o the i n n e r cutaneous anal r e g i o n through a s h o r t duct. Inputs t o these r e s e r v o i r s are the s e c r e t i o n s o f t h e glands o f the sac w a l l s and desquamated c e l l s from the sac e p i dermis . H i s t o l o g y r e v e a l s t h a t the fox a n a l sac t i s s u e s resemble those o f the dog i n c o n t a i n i n g mainly c o i l e d , t u b u l a r , apocrine glands, sebaceous glands being l a r g e l y confined t o the w a l l s o f the ducts (23,24) . I n c o n t r a s t , the anal sac w a l l s o f the c a t , f


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l i o n and t i g e r c o n t a i n plaques of sebaceous t i s s u e so t h a t these species produce l i p i d - r i c h a n a l sac s e c r e t i o n s (25,26), w h i l e the a n a l sac s e c r e t i o n s o f such mustelids as the s t r i p e d skunk, mink and p o l e c a t d i f f e r f u r t h e r i n c o n t a i n i n g s u b s t a n t i a l q u a n t i t i e s of v o l a t i l e organo-sulfur compounds (27,28). Our observations on mink anal sac t i s s u e using e l e c t r o n microscope techniques suggest t h a t , i n t h i s s p e c i e s , these substances probably d e r i v e from s u l f u r - r i c h granules present i n the numerous apocrine glands, which together w i t h sebaceous t i s s u e , make up a g l a n d u l a r complex around the neck o f the sac (Albone, F l o o d , Heap and Z i n k e v i c h , i n preparation). A f u r t h e r f a c t about the anal sac o f the r e d f o x (and very probably o f other c a r n i v o r e s ) i s t h a t i t supports an abundant m i c r o f l o r a (29^30/3^) This means t h a t a l l i n p u t s t o the sac from i t s w a l l s are subjected t o the a c t i v i t i e s o f a m i c r o b i a l ecosystem which s u b s t a n t i a l l y determines the nature o f the s e c r e t i o n f i n a l l y voided by the f o x . Only c e r t a i n types o f micro organism have been detected i n the f o x a n a l sac i n s u b s t a n t i a l numbers. T h i s , together w i t h the degree o f u n i f o r m i t y o f the aerobic m i c r o f l o r a a t the genus l e v e l observed from f o x t o f o x suggests a s t r u c t u r e d a n a l sac micro-ecosystem. The r e s u l t s presented i n Table I r e v e a l t h a t the aerobic sac m i c r o f l o r a i s dominated by s t r e p t o c o c c i and Proteus spp., w h i l e other organisms, such as c o l i f o r m s and gut l a c t o b a c i l l i are l a r g e l y absent, even though the sac i s s u f f i c i e n t l y c l o s e t o t h e anus t o permit passage o f f e c a l f l o r a i n t o the sac. Staphylo c o c c i , common s k i n micro-organisms i n many animals, were not found i n the sac. Of 96 a n a l sac s e c r e t i o n samples from 28 foxes examined, both Proteus spp. and s t r e p t o c o c c i were abundant ( :> 10 organisms/ml) i n 49 samples, and e i t h e r Proteus spp. o r s t r e p t o c o c c i were abundant i n 43 samples. Only i n 4 samples were n e i t h e r genera abundant. F u r t h e r , each genus was represented predominantly by one species (S. f a e c a l i s and P. m i r a b i l i s ) . S t r e p t o c o c c i were detected i n 89 samples (Proteus spp i n 87) and of these, i n 57 samples, s t r e p t o c o c c i were represented by a s i n g l e species (80 samples f o r Proteus) and i n 31 by two species (7 samples f o r P r o t e u s ) . There were no obvious c o r r e l a t i o n s i n the aerobic m i c r o f l o r a a t the species l e v e l w i t h season, age group, sex or i d e n t i t y o f the foxes samples. The s e l e c t i v e nature o f the anal sac microenvironment was r e v e a l e d by the f a i l u r e o f a number o f attempts t o e s t a b l i s h E s c h e r i c h i a c o l i i s o l a t e d from f o x feces as a r e s i d e n t species i n an a n a l sac. F o r example, Ε.coli i n j e c t e d i n t o a f o x a n a l sac t o g i v e an i n i t i a l p o p u l a t i o n o f 8 χ 1θ9 organisms/ml were reduced to 12% o f t h i s l e v e l i n 2 days, t o 3% i n 4 days and were undetectable a f t e r 15 days, w h i l e s t r e p t o c o c c i and Proteus gPP«-j_^ same ^ sample maintained populations i n the ranges 10 -10 and 10 -10 organisms^ml r e s p e c t i v e l y . S i m i l a r l y , a p o p u l a t i o n o f Ε.coli (1.8 χ 10 organisms i n 50 μΐ) incubated a n a e r o b i c a l l y i n v i t r o i n 250 μΐ u n s t e r i l i z e d anal sac s e c r e t i o n was e l i m i n a t e d a f t e r #

nt h e


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24 hours. T h i s e f f e c t i s a l s o r e f l e c t e d by a comparison o f anal sac and f e c a l m i c r o f l o r a . Of 18 f e c a l samples taken from 9 f o x e s , s t r e p t o c o c c i were detected i n 17 and were abundant ( *>* 10^ organisms/ml) i n 12, w h i l e Proteus spp. were observed i n only 7 samples and were abundant i n none and, c o n v e r s e l y , Ε.coli were observed i n 14 samples and were abundant i n 14. (Compare Table I)


Table I .

Occurrence o f a e r o b i c b a c t e r i a ( f a c u l t a t i v e anaerobes) i n red fox a n a l sac s e c r e t i o n s (29,30,31)

Organism

96 Samples from 28 foxes examined Detected (no.samples)

Abundant^ (no. samples)

Streptococcus f a e c a l i s Streptococcus faecium Streptococcus u b e r i s Streptococcus m i t i s

83 22 9 8

60 6 1 2

Proteus v u l g a r i s Proteus m i r a b i l i s Proteus r e t t g e r i

4 81 9

1 71 2

Escherichia c o l i K l e b s i e l l a spp./other c o l i f o r m s S e r r a t i a spp. Mi cro coc eus spp. Staphylococcus spp. N e i s s e r i a spp. B a c i l l u s spp. Diplococcus spp. L a c t o b a c i l l u s spp.

26 13 5 5 10 1 13 6 6

1 0 0 0 0 0 0 0, ( b )

o

v

( b )

(a) "> 10^ organisms/ml (b) L a c t o b a c i l l i were sought i n 37 samples from 15 foxes o n l y . L a c t o b a c i l l i were only detected i n foxes l e s s than 3 months o l d (4 o u t o f 6 foxes i n t h i s age group) Fox a n a l sac s e c r e t i o n samples were a l s o examined anaerob i c a l l y using pre-reduced, a n a e r o b i c a l l y s t e r i l i z e d media (32), t a k i n g r i g i d p r e c a u t i o n s t o avoid exposure t o the a i r . Anaerobes were c l a s s i f i e d by o b t a i n i n g API 20A biochemical p r o f i l e s (Analytab Products Inc.) and employing s i n g l e l i n k a g e c l u s t e r a n a l y s i s (33) t o group s i m i l a r organisms w i t h s e l e c t e d API 20A reference species (31). Some 127 d i f f e r e n t anaerobic i s o l a t e s from 66 s e c r e t i o n s from 18 foxes y i e l d e d predominantly C l o s t r i d i a (Table I I , column A ) . An examination o f 107 f u r t h e r i s o l a t e s from 37 s e c r e t i o n s from 6 foxes, c o l l e c t e d under the most r i g o r o u s anaerobic c o n d i t i o n s and employing a s p e c i a l l y designed c o n t a i n e r to exclude a i r and maintain a carbon d i o x i d e environment around


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the hind-quarters o f the sedated f o x during sample c o l l e c t i o n , revealed the presence o f o t h e r , more oxygen-sensitive anaerobes (Table I I , column B ) . Anaerobic p o p u l a t i o n determinations are s u b j e c t t o e r r o r because o f the u n c e r t a i n t y o f percentage r e c o v e r a b i l i t y i n primary c u l t u r e . From microscopic counts, s t r i c t anaerobic p o p u l a t i o n s were estimated t o be comparable w i t h those o f aerobes, f r e q u e n t l y i n the range o f ΙΟ^-ΙΟ organisms/ ml s e c r e t i o n .


10

Table I I . Occurrence o f s t r i c t anaerobes i n r e d f o x anal sac s e c r e t i o n s (31). I s o l a t e s c l u s t e r i n g a t s i m i l a r i t y c o e f f i c i e n t 0.75 on the b a s i s o f API 20A biochemical prο f i l e s, morphology and Gram s t a i n r e a c t i o n (see t e x t ) Reference organism Clostridium perfringens C l o s t r i d i u m ramosum C l o s t r i d i u m sporogenes C l o s t r i d i u m bifermentans Eubacterium lentum Bifidobacterium e r i k s o n i i Fusobacterium nucleatum Fusobacterium negrogenes Bacteroides f r a g i l i s f r a g i l i s Peptostreptococcug anaerobius unclustered

A ^ (no.isolates)

B ^ (no.isolates)

50 16 27 21 13 0 0 0 0 0 0

37 0 4 1 4 2 6 17 20 7 (O 9

(a) From 66 s e c r e t i o n s from 18 foxes, employing anaerobic techniques. (b) From 37 s e c r e t i o n s from 6 foxes, employing anaerobic techniques i n c l u d i n g an a i r - f r e e sampling environment, see text. (c) C l u s t e r i n g a t lower s i m i l a r i t y w i t h B i f i d o b a c t e r i u m e r i k s o n i i (6) and Peptostreptococcus anaerobius ( 3 ) . P r e l i m i n a r y observations i n d i c a t e t h a t the f o x anal sac may generate h i g h l y reducing c o n d i t i o n s , w i t h redox p o t e n t i a l s down to -400mV. A redox p o t e n t i a l o f -200mV has been recorded i n the cecum o f the mouse (34). I n the a n a l sac, i t appears t h a t f a c u l t a t i v e anaerobic organisms, such as s t r e p t o c o c c i and Proteus spp. create and maintain an anaerobic environment i n which s t r i c t anaerobes ( f r e q u e n t l y a c t i v e odor producers) can grow. In accord w i t h t h i s , chemical s t u d i e s so f a r undertaken on the a n a l sac s e c r e t i o n s o f the r e d f o x and other c a r n i v o r e s i n d i c a t e t h a t major low molecular weight components present are g e n e r a l l y commonly encountered products o f m i c r o b i a l a c t i v i t y . V o l a t i l e f a t t y a c i d s are major c o n s t i t u e n t s o f the a n a l sac s e c r e t i o n s o f the r e d f o x and the l i o n (29,35), the coyote (36),



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the domestic dog (36), and the mink (37). They are a l s o present i n the anal pocket s e c r e t i o n of the Indian mongoose (38) and i n the p e r i n e a l gland s e c r e t i o n o f the guinea p i g (39). In the cases of the red fox and of the Indian mongoose, evidence has been advanced to confirm t h e i r m i c r o b i a l o r i g i n . These same a c i d s occur i n a v a r i e t y of other mammalian scent sources, i n c l u d i n g the vagina o f primates, where a m i c r o b i a l o r i g i n has a l s o been s u b s t a n t i a t e d and a communicatory r o l e discussed (40), as w e l l as i n the v a g i n a l f l u i d s of other s p e c i e s , such as mink (41). Thus, i n the case of fox anal sac s e c r e t i o n , an inoculum of f r e s h anal sac s e c r e t i o n (100 μΐ) c o l l e c t e d a n a e r o b i c a l l y and incubated a n a e r o b i c a l l y i n pre-reduced Robertson's cooked meat medium (10 ml) a t 37 C f o r 48 hours y i e l d e d v o l a t i l e f a t t y acids a t concentrations ( a c e t i c a c i d , 170 mM; t o t a l C^-Cg a c i d s , 95 mM, by gas chromatography) comparable with, or i n excess of, those encountered i n anal sac s e c r e t i o n s . 152 s t r i c t l y anaerobic b a c t e r i a l i s o l a t e s from 78 anal sac s e c r e t i o n s from 19 foxes were examined f o r v o l a t i l e f a t t y a c i d production under s i m i l a r c o n d i t i o n s . Anaerobes of a l l genera produced some or a l l the v o l a t i l e f a t t y a c i d s noted i n anal sac s e c r e t i o n , c e r t a i n C l o s t r i d i a and e u b a c t e r i a being the most a c t i v e producers, although great v a r i a t i o n s i n production were noted between d i f f e r e n t i s o l a t e s from the same genus. In the sac i t s e l f , m i c r o b i a l production of v o l a t i l e f a t t y a c i d s was confirmed by i r r i g a t i n g the sac with 1% aqueous sodium h y p o c h l o r i t e (containing 16.5% sodium c h l o r i d e ) , followed with p h y s i o l o g i c a l s a l i n e and then by f i l l i n g the sac with a n t i b i o t i c (10% a m p i c i l l i n / 0 . 5 % t e t r a c y c l i n i n p h y s i o l o g i c a l s a l i n e ) . Neither b a c t e r i a nor v o l a t i l e f a t t y a c i d s were detected i n the sac f o r i n excess o f 3 days subsequently. Other anal sac c o n s t i t u e n t s which are commonly encountered products of m i c r o b i a l a c t i v i t y i n c l u d e trimethylamine, noted i n the anal sac s e c r e t i o n s of the red fox (42), coyote and domestic dog (36), and the aromatic a c i d s p h e n y l a c e t i c a c i d and 3-phenylp r o p i o n i c a c i d (and r e l a t e d phenolic a c i d s ) , together with the diamines p u t r e s c i n e and cadaverine as w e l l as ammonia i n the anal sac s e c r e t i o n s of the red fox and the l i o n (25,29,35). Indole has a l s o been noted. The lower molecular weight l i p i d s o f l i o n anal sac s e c r e t i o n i n c l u d e many substances expected as h y d r o l y s i s products of sebaceous l i p i d s (25) . Red fox anal sac s e c r e t i o n a l s o e x h i b i t s an anomalous f r e e amino-acid composition with 5-aminovaleric a c i d predominating (43). The p o s s i b i l i t y that the s u l f u r - c o n t a i n i n g v o l a t i l e s present i n mustelid anal sac secre t i o n s are of m i c r o b i a l o r i g i n i s a t present under i n v e s t i g a t i o n i n our l a b o r a t o r i e s . f

Fermentative Scent Sources: II) Environmental;

Carrion

C a r r i o n , which may be detected a t l e a s t i n p a r t by odor cues, possesses considerable a t t r a c t i o n f o r c a r n i v o r e s , whether as a



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food source or as a s i t e f o r scent marking. M i c r o b i a l p u t r e f a c t i o n a r i s e s i n the carcass of a dead animal l a r g e l y as the r e s u l t o f b a c t e r i a l p e n e t r a t i o n of the gut a f t e r r e s i d u a l t i s s u e a n t i m i c r o b i a l a c t i v i t y has been l o s t and when anoxic c o n d i t i o n s have been e s t a b l i s h e d as the r e s u l t o f continued t i s s u e metabo l i s m i n the absence of oxygen. S t u d i e s i n t h i s area are few and have been undertaken p r i n c i p a l l y i n the context o f f o r e n s i c s c i e n c e . A v a l u a b l e review o f t h i s work i s p r o v i d e d by Corry (44), who r e p o r t s t h a t , although many d i f f e r e n t anaerobes r e s i d e i n the i n t e s t i n e , o n l y a few groups have been i m p l i c a t e d so f a r as major c o l o n i z e r s of corpses d u r i n g p u t r e f a c t i o n . The most important o f these i s C l o s t r i d i u m p e r f r i n g e n s , a vigorous s a c c h a r o l y t i c , l i p o l y t i c and p r o t e o l y t i c organism which i s a l s o commonly r e s i d e n t i n the fox anal sac. In a d d i t i o n , v o l a t i l e s o f the type i d e n t i f i e d i n a n a l sac s e c r e t i o n s are commonly produced when gut microorganisms are incubated a n a e r o b i c a l l y i n p r o t e i n - r i c h media. Thus, the low molecular weight m e t a b o l i t e s produced by C l o s t r i d i a i n c l u d e 5-aminovaleric a c i d , the v o l a t i l e f a t t y a c i d s and the amines i d e n t i f i e d i n anal sac s e c r e t i o n s (45,46,47). P u t r e f i e d animal matter has formed the b a s i s f o r coyote a t t r a c t a n t s o f p o s s i b l e value i n p e s t c o n t r o l programs. Thus, a p u t r e f i e d f i s h f o r m u l a t i o n has been used as a coyote l u r e and, more r e c e n t l y , a t t e n t i o n has been d i r e c t e d t o a fermented aqueous suspension of chicken whole-egg powder, developed i n i t i a l l y as an a t t r a c t a n t f o r f l i e s (48). The odor components of t h i s m a t e r i a l have been subjected to d e t a i l e d chemical a n a l y s i s ky B u l l a r d e t a l . (49) and are r e p o r t e d t o i n c l u d e v o l a t i l e f a t t y a c i d s (77% t o t a l ; 13 a c i d s i d e n t i f i e d ) , bases (13% t o t a l , mainly trimethylamine, 9 amines i d e n t i f i e d ) , and headspace v o l a t i l e s , i n c l u d i n g e s t e r s , aldehydes, ketones, a l c o h o l s , a l k y l aromatics, terpenes and s u l f u r compounds (10% t o t a l , 76 compounds i d e n t i f i e d ) . Based on these d a t a , a s y n t h e t i c m i x t u r e , " s y n t h e t i c fermented egg" has been formulated, composed l a r g e l y of a mixture of ten v o l a t i l e f a t t y a c i d s (81%), together w i t h a d i v e r s e range of amines and other compounds (50). T h i s mixture was found to be as a t t r a c t i v e t o coyotes as the fermented p r e p a r a t i o n i t s e l f . The v o l a t i l e f a t t y a c i d component alone was found to e x h i b i t subs t a n t i a l coyote a t t r a c t a n c y a l s o (50,51) . As a f u r t h e r example o f the a t t r a c t a n c y o f m i c r o b i a l products an unfortunate case i s quoted i n the medical l i t e r a t u r e (52) o f a woman s u f f e r i n g from a s k i n c o n d i t i o n which, as the r e s u l t o f secondary i n f e c t i o n , emitted obnoxious odors which caused her t o be molested by dogs. In a d i f f e r e n t c o n t e x t , Amoore and F o r r e s t e r (5_3) quoted Linnaeus' o b s e r v a t i o n t h a t the domestic dog i s a t t r a c t e d by the odor of the p l a n t Chenopodium v u l v a r i a , the leaves o f which are r i c h (400 ppm) i n trimethy1amine. Mammalian and Environmental Fermentative Scent Sources Compared Even on the b a s i s o f present l i m i t e d knowledge, chemical and



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m i c r o b i o l o g i c a l s i m i l a r i t i e s a r e emerging f o r the r e d fox between a mammalian scent source, the anal sac, and an important e n v i r o n mental scent source, c a r r i o n . T h i s i s not t o say t h a t these scents are not d i s t i n g u i s h a b l e t o the f o x . Fundamental d i f f e r ences remain t o the extent t h a t the anal sac fermentation i s c o n t r o l l e d and l i m i t e d by the l i v i n g mammal. The o b s e r v a t i o n o f s t r u c t u r e i n the a n a l sac micro-ecosystem seems t o suggest the o p e r a t i o n o f s e l e c t i v e pressures and so t o imply the e x i s t e n c e o f s p e c i f i c b i o l o g i c a l f u n c t i o n s o f the a n a l sac o f adaptive value f o r the fox. Superimposed on the general a t t r a c t a n c y which fermentative scent sources e x e r t f o r c a n i d s , the mammal can convey more s p e c i f i c i n f o r m a t i o n t o the degree t h a t the a n a l sac scent source responds t o the p h y s i o l o g i c a l and s o c i a l circumstances o f the mammal. Although very l i t t l e research has y e t been conducted on any such scent source i n t h i s context, two suggestive examples o f such processes i n other species have been documented. Thus, through hormonal r e g u l a t i o n o f s u b s t r a t e a v a i l a b i l i t y , the rhesus monkey i s able t o r e g u l a t e the p r o d u c t i o n o f v o l a t i l e compounds by i t s v a g i n a l m i c r o f l o r a so t h a t m i c r o b i a l scent p r o d u c t i o n from t h a t source r e f l e c t s the r e p r o d u c t i v e s t a t e o f the monkey (16). And, i n the r a t , maternal pheromone, the scent which a t t r a c t s the pups t o the mother, a r i s e s from the a c t i o n o f b a c t e r i a on food i n the cecum. This i s r e g u l a t e d by high p r o l a c t i n l e v e l s which s t i m u l a t e the l a c t a t i n g r a t t o high food and water consumption and l e a d t o the p r o d u c t i o n and emission o f excess cecotrophe, thus p r o v i d i n g the chemical s i g n a l (54) . The p o s s i b i l i t y o f mammalian fermentative scent sources a c q u i r i n g i n d i v i d u a l o r group r e c o g n i t i o n value has a l s o been d i s c u s s e d i n r e l a t i o n t o the r e d fox and the I n d i a n mongoose, although the experimental evidence a t present a v a i l a b l e i n support of t h i s o c c u r r i n g i n these species i s n o t strong (5,35,55). The s i g n a l l i n g f u n c t i o n ( s ) o f a n a l sac s e c r e t i o n i n the l i f e of the f o x i n i t s n a t u r a l environment remain(s) u n c l e a r , r e f l e c t ing the p a u c i t y o f i n f o r m a t i o n which e x i s t s on the chemical ecology o f w i l d mammalian s p e c i e s . As w i t h u r i n e , i t probably conveys a v a r i e t y o f messages i n a v a r i e t y o f circumstances (56). P r e l i m i n a r y f i e l d observations (_4) i n d i c a t e t h a t the s e c r e t i o n may be deposited w i t h feces o r t h a t i t may be emitted without d e f e c a t i o n when the f o x i s alarmed. Anal sac evacuation has been observed a t t e r r i t o r i a l boundaries f o l l o w i n g e v i c t i o n o f an i n t r u d e r . However, an exhaustive a n a l y s i s o f the s i g n a l l i n g r o l e of t h i s s e c r e t i o n has y e t t o be undertaken. Our l a b o r a t o r y s t u d i e s on the b e h a v i o r a l response o f the r e d f o x t o a n a l sac s e c r e t i o n have, l i k e those o f Doty and Dunbar w i t h beagle a n a l sac s e c r e t i o n (57), proved o f l i m i t e d v a l u e . T h i s i s not unexpected when the gross nature o f the responses assessed (frequency and d u r a t i o n o f i n v e s t i g a t i o n ) i n a r t i f i c i a l surroundings are compared w i t h the complexity o f the r e l a t i o n s h i p o f a w i l d mammal to i t s n a t u r a l environment. I d e a l l y , one would i n v e s t i g a t e the


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d e t a i l e d e f f e c t o f chemical s i g n a l s on the e n t i r e b e h a v i o r a l r e p e r t o i r e o f w i l d animals. F i e l d s t u d i e s on the s i g n i f i c a n c e o f scent marks i n the l i f e o f w i l d mammals remain few. Notable examples are those o f Peters and Mech on the wolf (58) and of Henry on the r e d f o x (56).


Behavioral

studies

Basing our methods on those employed by L i n h a r t and Knowlton (59) using fermented egg product as a coyote a t t r a c t a n t , we have s t u d i e d the response o f the f o x t o fermented products r e l a t e d i n some measure t o anal sac s e c r e t i o n . A d e t a i l e d account o f t h i s work w i l l be p u b l i s h e d s h o r t l y (Macdonald, Hough, B l i z a r d and Perry, i n preparation). Fermented egg product i s produced by exposing an aqueous suspension o f chicken whole-egg powder t o the a i r a t room temperature f o r 7 t o 14 days. The n u t r i e n t i s c o l o n i z e d by micro-organisms from the a i r , and these b r i n g about fermentation. As w e l l as being u n c o n t r o l l e d , t h i s method o f i n o c u l a t i o n excludes s t r i c t anaerobes which are among the most e f f e c t i v e odor producers. I n our i n i t i a l experiments, we have compared the a t t r a c t a n c y o f a f o x t i s s u e e x t r a c t (FE), incubated a n a e r o b i c a l l y w i t h an inoculum o f f r e s h , a n a e r o b i c a l l y c o l l e c t e d a n a l sac s e c r e t i o n ( F E I ) , w i t h (a) a f o x t i s s u e e x t r a c t c o n t r o l , not so i n o c u l a t e d and incubated (FEC); (b) r e d f o x ($£) u r i n e ; (c) d i s t i l l e d water. FEI possesses the advantages o f being r e a d i l y prepared i n bulk f o r f i e l d t e s t i n g w h i l e m i r r o r i n g , probably more c l o s e l y than other r e a d i l y a v a i l a b l e fermentations, features o f f o x a n a l sac s e c r e t i o n s . The f o x t i s s u e e x t r a c t medium was prepared by simmering f r e s h r e d fox meat (1.2 kg h i n d l e g m u s c l e / l i v e r ) w i t h water f o r one hour, d r a i n i n g the e x t r a c t , a d j u s t i n g i t s pH t o 8.2 and f i l t e r i n g . To 1000 ml o f the r e s u l t i n g l i q u i d , 20 ml c y s t e i n e / s u l f i d e s o l u t i o n (1.5 g L - c y s t e i n e hydrochloride/1.5 g Na^S^H^O i n 100 ml aqueous s o l u t i o n ) were added f o l l o w e d by 1 g L-methionine, 5 g NaCl and 10 g D i f c o Bacto-peptone t o y i e l d the f i n a l f o x t i s s u e e x t r a c t medium. Incubations were conducted a t 37 f o r 60 hours. Anaerobic i n c u b a t i o n s s i m i l a r l y prepared from other media using a n a l sac i n o c u l a , were a l s o t e s t e d . These media were Robertson's meat broth (RM), c a s e i n a c i d hydrolysate medium (CHY) and an egg y o l k medium (EY) ( f r e s h egg y o l k / g l u c o s e / D i f c o b r a i n heart i n f u s i o n b r o t h ; 5/2/100 w/w/w). A f t e r i n c u b a t i o n , a l l fermentations were stopped by the a d d i t i o n o f excess s o l i d sodium c h l o r i d e , and maintained a t 4°C u n t i l r e q u i r e d f o r t e s t i n g . Tests were conducted i n a small deciduous woodland where the movements o f many o f the r e s i d e n t foxes had e a r l i e r been establ i s h e d by r a d i o - t r a c k i n g (_4) . Probably a t l e a s t e i g h t a d u l t foxes (2 male, 6 female) were using the experimental area n i g h t l y . From d i r e c t observations and f i e l d s i g n s , the most h e a v i l y used t



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fox paths through the woodland were i d e n t i f i e d and these were used i n the subsequent t r i a l s . V e g e t a t i o n was c l e a r e d from two c i r c u l a r areas (60 cm diameter) on opposite s i d e s o f , and a d j o i n i n g , the s e l e c t e d fox path. T h i s process was repeated a t i n t e r v a l s o f 5 m u n t i l s i x p a i r s o f such c i r c l e s were prepared. I n each case, l e a f l i t t e r was removed and the top s o i l f i n e l y s i f t e d u n t i l i t formed a s u f f i c i e n t l y deep l a y e r t o r e l i a b l y d e t e c t i n d i v i d u a l f o o t p r i n t s . In a d d i t i o n , f u r t h e r s i f t e d areas were prepared spanning the path between each c i r c l e p a i r . In the l a t e afternoon o f the f i r s t experimental day ( A p r i l ) , a s m a l l cane b e a r i n g a c i r c u l a r f i l t e r paper (4.25 cm) was p o s i t i o n e d i n the c e n t e r o f each earthen c i r c l e , standing a t a height o f some 15 cm and an a l i q u o t (1 ml) o f a g i v e n odorant was dropped on t o the f i l t e r paper. On each subsequent day a t a s i m i l a r time the earthen c i r c l e s were examined f o r f o o t p r i n t s , the canes were r e p l a c e d by cleaned canes b e a r i n g f r e s h f i l t e r paper and a new odorant, and the s o i l c a r e f u l l y s i f t e d a g a i n . The d i s t r i b u t i o n o f odorant along the path ( s p a c i a l d e n s i t y and l o c a t i o n on v i s u a l l y conspicuous objects)resembled the d i s t r i b u t i o n o f scent (urine) marks commonly produced by a fox i n t h i s environment. Each experiment compared f o u r odorants i n each o f the s i x p o s s i b l e b i n a r y combinations, one combination f o r each c i r c l e p a i r . Odorants were thus t e s t e d i n p a i r s , randomizing t h e sequence o f p r e s e n t a t i o n o f each p a i r along the path from n i g h t to n i g h t . The experiment was continued u n t i l every p a i r e d combination o f odors had been exposed t o foxes f o r 10 n i g h t s , d i s c o u n t i n g n i g h t s when an absence o f t r a c k s on the s i f t e d s e c t i o n s between the c i r c l e s r e v e a l e d t h a t no foxes had f o l l o w e d t h a t path t h a t n i g h t . Thus, the s i x p o s s i b l e p a i r s o f f o u r odorants were compared 10 times. A d i s c u s s i o n o f the s t a t i s t i c a l a n a l y s i s o f p a i r e d comparisons o f t h i s type has been g i v e n by Brown (60). In the f i r s t experiment, subsequently repeated i n t r i p l i c a t e w i t h s i m i l a r r e s u l t s , fox t i s s u e e x t r a c t i n c u b a t i o n (FEI), f o x t i s s u e e x t r a c t c o n t r o l (FEC), fox u r i n e and water were compared. FEI was found t o e l i c i t a s i g n i f i c a n t l y g r e a t e r response than e i t h e r fox u r i n e o r the two c o n t r o l s . T h i s i s o f c o n s i d e r a b l e i n t e r e s t as q u a l i t a t i v e o b s e r v a t i o n s on fox behavior have a l r e a d y i n d i c a t e d the g r e a t a t t r a c t a n c y o f a l i e n fox u r i n e ( 4 ) . T h i s r e s u l t was i n d i c a t e d i n two ways. F i r s t , a simple nominal ranking based on the number o f n i g h t s odorants were v i s i t e d over the e n t i r e experimental sequence i n d i c a t e d t h a t FEI was v i s i t e d on s i g n i f i c a n t l y more n i g h t s than FEC o r water, and t h a t u r i n e was v i s i t e d on s i g n i f i c a n t l y more n i g h t s than water. Thus, i n a t y p i c a l experimental sequence, FEI was v i s i t e d on 21 n i g h t s ; u r i n e , 16; FEC, 11; y p t e r , 7 (maximum s c o r e , 30). These d i f f e r e n c e s were not random (}( , 8.05; d f , 3; p< 0.05) . In order t o rank the a t t r a c t a n c y on an o r d i n a l s c a l e , a s c a l i n g f a c t o r based on the f o o t p r i n t count w i t h i n 30 cm o f an odorant was employed. S c a l e d mean scores were obtained f o r each


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odorant on each n i g h t using a m a t r i x o f d i f f e r e n c e scores f o r each odor (60). O v e r a l l s c a l e d mean scores f o r each odorant (the means o f the n i g h t l y s c a l e d mean s c o r e s , taken over the t e n nights) a r e given i n Table I I I . Randomized b l o c k a n a l y s i s o f v a r i a n c e showed t h a t i n s p i t e o f v a r i a t i o n s between i n d i v i d u a l n i g h t s , d i f f e r e n c e s between scores were s t a t i s t i c a l l y s i g n i f i c a n t . Table I I I . A t t r a c t a n c y t o odorants Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2017 | http://pubs.acs.org Publication Date: February 2, 1978 | doi: 10.1021/bk-1978-0067.ch005

Experiment

O v e r a l l s c a l e d mean scores f o r odorant FEI

Red f o x ; f o o t p r i n t number 1.55 F e r r e t ; frequency 0.53 Ferret; t o t a l d u r a t i o n (sec) 3.45 F

E

(a) [ c x ]

(see t e x t )

3 / 2

7

=

3

'

5 1

(

P

=

urine 0.38 1.35 14.75 °-

FEC

water

-0.6 -1.05

-1.3 -0.83

-9.15

-9.05

Randomized b l o c k a n a l y s i s o f variance a error MS F 15.41 12.94 1316.97

1.73 1.39

8.91 9.28

38.23

34.45

0 1 )

The a n a l y s i s o f t h e s i g n i f i c a n c e o f d i f f e r e n c e s between p a i r s of s c a l e d mean scores p r e s e n t s s e r i o u s s t a t i s t i c a l problems which have been overcome using Brown's method (60). F o r a f u l l d i s c u s s i o n , see Macdonald, Hough, B l i z a r d and P e r r y , i n p r e p a r a t i o n . The r e s u l t s a l l o w the f o l l o w i n g preference rank t o be concluded FEI^

f o x u r i n e > FEC s

water

The measure o f a t t r a c t a n c y used was very crude and, i n order to begin t o c l a r i f y the s p e c i e s s i g n i f i c a n c e o f these r e s u l t s , we examined the responsesof a f e r r e t , Mustela f u r o , t o these substances. Odorants were presented t o a male f e r r e t i n p a i r s (randomized p o s i t i o n and order d u r i n g 60 (10 minute) t r i a l s . A l i q u o t s (1 ml) o f each odorant were presented on two b l o c k s covered w i t h f i l t e r paper. T r i a l s were continued u n t i l t e n r e p l i c a t e s o f each o f the s i x b i n a r y combinations o f the f o u r odorants had been performed. Duration and frequency o f odorant i n v e s t i g a t i o n were noted. The f e r r e t showed c o n s i d e r a b l e i n t e r e s t i n the b l o c k s bearing FEI and f o x u r i n e , r e l a t i v e t o those b e a r i n g FEC and water, repeatedly v i s i t i n g these b l o c k s d u r i n g each t r i a l and s l i t h e r i n g across them, sometimes u r i n a t i n g and probably a l s o l e a v i n g g l a n d u l a r s e c r e t i o n s . The o v e r a l l s c a l e d mean scores o f both the v a r i a b l e s measured are g i v e n i n Table I I I . The r e s u l t s o f analyses of v a r i a n c e o f both these measures i n d i c a t e s h i g h l y s i g n i f i c a n t d i f f e r e n c e s between these scores (p < 0.001). Thus, as w i t h the f i e l d t r i a l s w i t h the f o x , f o x u r i n e and FEI were p r e f e r r e d t o FEC and water, but the ranking was d i f f e r e n t .


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An a n a l y s i s as p r e v i o u s l y gives the f o l l o w i n g preference based on frequency o f i n v e s t i g a t i o n .

89

rank


Fox u r i n e > FEI > FEC = water Although these f i n d i n g s are p r e l i m i n a r y and f u r t h e r s t u d i e s are c u r r e n t l y i n progress, they do i n d i c a t e t h a t FEI i s very a t t r a c t i v e t o foxes (although they do not i n d i c a t e the f u n c t i o n a l s i g n i f i c a n c e o f t h i s a t t r a c t i o n ) and t h a t t h i s a t t r a c t i o n a r i s e s as the r e s u l t o f anaerobic i n c u b a t i o n . The d i f f e r e n c e i n ranking noted i n the experiment w i t h the f e r r e t , i f confirmed by more extensive t e s t i n g , would i n d i c a t e t h a t the a t t r a c t a n c y has a degree o f species s p e c i f i c i t y and t h i s , i n t u r n , would open the way t o f u r t h e r i n v e s t i g a t i o n s o f both academic and a p p l i e d importance on the b i o l o g i c a l f u n c t i o n s o f scent s i g n a l s . I n t h i s context, i t i s o f i n t e r e s t t o note t h a t s i m i l a r p r e l i m i n a r y f i e l d s t u d i e s using anaerobic i n c u b a t i o n and unincubated c o n t r o l s o f the other media (RM, CHY, EY) have revealed only weak f o x a t t r a c t a n c y compared w i t h the fox t i s s u e e x t r a c t i n c u b a t i o n . I s t h i s because the fox t i s s u e e x t r a c t i n c u b a t i o n possesses some f e a t u r e i n common w i t h i n c u b a t i o n s o c c u r r i n g i n the f o x e s own anal sac? These phenomena r e q u i r e f u r t h e r c o n f i r m a t i o n and c l a r i f i c a t i o n . However, l a b o r a t o r y t e s t s so f a r undertaken w i t h four a d u l t male foxes using an olfactometer t o measure frequency and d u r a t i o n o f v i s i t s t o odor p o r t s a r e broadly i n accord w i t h these f i e l d t r i a l s (Macdonald, Hough, B l i z a r d and P e r r y , i n preparation). 1

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Bacterial Action and Chemical Signalling in the Red Fox (Vulpes

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