Journal ~f Chemical Ecology, VoL 22, No. 2. 1996
ANAL GLAND SECRETION OF EUROPEAN MOLE: VOLATILE CONSTITUENTS AND SIGNIFICANCE IN TERRITORIAL MAINTENANCE
CHANTAL
KHAZANEHDARI, and JOHN
ALAN
J. B U G L A S S ,
S. W A T E R H O U S E *
Enlqronmental Science Research Centre Anglia Polytechnic University East Rd., Cambridge. CBt IPT. U.K~ (Received June 6, 1995; accepted October 16, 1995)
Abstract--The chemical composition of the volatile components of the anal gland secretion of mature and immature moles of both sexes was examined by gas chromatography and mass spectrometry. The compounds in the secrelion of adult males appear to vary little throughout the year and are similar to those from the adutt female outside the breeding season. The secretions are dominated by C5-C~o carboxylic acids. Female glands regress as they enter proestms, accompanied by profound changes in the chemical composition of the secretion with early disappearance of the carboxylic acids. In juvenile moles, the compostion of the secretion changes as the animal matures, with carboxylic acids becoming dominant only as the animal reaches maturity. Anal gland secretion probably plays an important role in territorial maintenance. Key Words--Moles, Talpa europaea, anal glands, scent marking, territoriality, semiochemical, proestrus, carboxylic acids.
INTRODUCTION F o r m o s t o f the y e a r t h e E u r o p e a n m o l e (Talpa europaea) l i v e s a s o l i t a r y life in its o w n s u b t e r r a n e a n t u n n e l s y s t e m a n d a c t i v e l y a v o i d s c o n t a c t w i t h o t h e r m e m b e r s o f its o w n s p e c i e s . T h e h o m e r a n g e s o f f e m a l e s o r o f a m a l e a n d a f e m a l e m a y , h o w e v e r , o v e r l a p , a n d l e n g t h s o f t u n n e l m a y be u s e d in c o m m o n , M o l e s in o v e r l a p p i n g t e r r i t o r i e s are a w a r e o f e a c h o t h e r ' s e x i s t e n c e a n d m o v e *To whom correspondence should be addressed. 383 00984)33t/96/02fX~-0383~)9511/0~c~1996PlcnumPublishingCorpl~r,~li~n
384
KI-IAZANF~HI)ARI. BI.~(;LASS. AND WAFERI-IOIIN~:
ments, and they will organize their daily routine so as to avoid each other (Stone and Gorman, 1985; Gorman and Stone, 1990), The only time of the year when moles purposefully meet is during the mating season, when male moles will migrate into female territories in order to mate (Godfrey and Crowcroft, 1960). Studies by Gorman and Stone (1990) have suggested that the mole's efficient territorial maintenance is controlled at least in part by chemical signals produced by the secretion of specialized glands in the inguinal region of both sexes. These glands have been termed "'preputial glands" by some workers (Racey, 1978; Stone and Gom~an, 1985; Gorman and Stone, 1989, 1990), but we have recently shown that they are in fact anal glands (Clevedon Brown et al., 1994). We present results here that support the hypothesis that the secretion from the anal glands plays a central role in the territorial maintenance of the mole. Our evidence is based on studies on variations in glandular mass and the chemical composition of the volatile components of the glandular secretion throughout the year. METHODS AND MATERIALS
Moles used for both the anatomical and chemical studies were caught in lethal traps in Cambridgeshire and adjacent parts of Norfolk by a professional mole catcher (DRE Pest Control, Swavesey, Cambridgeshire). The carcasses were frozen immediately on collection for transport to the laboratory, where they remained frozen at - 2 0 ° C until dissection. Anal glands were separated from extraglandular material and weighed. The glandular secretion was released by crushing the gland, and the volatile components therein were trapped on an activated charcoal disc using a Gorb dynamic headspace apparatus as previously described (Buglass et al., 1990). The volatiles were eluted from the disc with 8 #1 of dichloromethane containing dihexyl sulfide (115 ng/txl as internal standard), and 1 p.l of the resulting solution injected into a Hewlett-Packard 5890 gas chromatograph equipped with a polydimethylsiloxane column (length, 15 m; ID, 0,25 ram) and connected to a Hewlett-Packard 5970 mass spectrometer detector. The temperature program was 35-240°C at a rate of 4°C/rain with a final hold at 240°C for 45 min. Compounds were identified from their mass spectra and also, in most cases, by comparison of retention times with those of authentic compounds (see Table 1 below). Male moles in breeding condition were distinguished from nonbreeding males by the great increase in size of the testes and epidydimus. Proestrus in the female was characterized by the presence of the vaginal opening, the increase in length and breadth of the reproductive tract resulting from growth and thickening of its wall, and vascularization of the uterine cornua (Hamson Matthews, 1935). We observed an increase in length of the uterovaginal canal to around
MOLE ANAL GLAND SECRETION
385
40-45 % of body length during proestrus, as opposed to a normal value of less than 20%, in line with the findings of Harrison Matthews (1935). As estrus itself lasts for only about 30 hr in the mote (Godfrey and Crowcroft, 1960L no attempt was made to distinguish between the proestrous and the estrous female.
RESULTS A N D D I S C U S S I O N
In East Anglia the mating season for moles occurs early in the year. Females approach estrus as early as January. The gestation period is four weeks and lactation follows tot a further four weeks (Deanesly, 1966: Godfrey and Crowcroft, 1960: Harrison Matthews, 1935): hence by February or March, females are likely to be pregnant or to be lactating. Additionally, we have observed a low incidence of second proestrus and presumably estrus in September. Preliminary results from our study of the changes in anal gland size during the year showed that the glands of males and females behave differently (Buglass et al., 1995). Male glands increase in weight during the breeding season, reaching a maximum value at the peak of the breeding season in February and March. This change is probably due to vascular development resulting from a surge in testosterone, which in the shrew serves to increase vascularity of sebaceous tissue (Pearson, 1946). The increase in size and weight of the gland is associated with a deepening of its color to dark red. In contrast, female glands undergo a reduction in size as the female enters estrus in January or February. In the majority of 14 females that were collected in January. the glands had regressed to such a degree that they were invisible to the naked eye. Female glands enlarge as the breeding period ends, but the average value drops sharply in May before climbing again during summer and fall. We explain this effect by the inclusion of juveniles in the catch tYom May onwards. The glands of juveniles are flaccid and less developed than those of the mature mole, and it is not until August or September that glands of moles born in that year become indistinguishable from those of mature animals. The results of an extension of this study, involving a greater number of moles, are shown in Figure 1. These results are in full agreement with our earlier findings. Samples of anal gland secretion from a total of 130 moles were analyzed by gas chromatography and mass spectrometry. For adult moles, chromatograms were obtained from secretion collection in each month of the year. A typical chromatogram from a nonbreeding male, a breeding male, a juvenile male, a nonbreeding female, a proestrous female, a pregnant female, a lactating female, a female approaching second estrus, and a juvenile female are shown in Figure 2A-I, respectively, Compounds are identified in Table 1. With the exception of the proestrous female, C s-Cto carboxytic acids, both straight and branched chain, tbrm a significant component of the secretions of
386
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FiG. t. Box and whisker plots showing the seasonal changes in the anal gland weight as a percentage of body weight for male and female moles. Numbers refer to number of moles in each sample.
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387
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FIG. 2, Gas chromatograms of volatile compounds from the anal gland secretions of moles: (A) nonbreeding male (October); (B) breeding male (April): (C) juvenile male (May); (D) nonbreeding female (October): (E) proestrous female (January); (F) pregnant female (February); (G) lactating female (March); (H) female approaching second estrus (September): (I) juvenile female (May). x = impurity; IS = internal standard (dihexyl sulfide).
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389
MOLE ANAL GLAND SECRETION TABLE [. COMPOUNDS IDENTIFIED IN ANAL GLAND SECRETION OF MOLE ( N u m b e r s in parentheses match those in Fig. 2.)
Carbo.~,lic acids Butanoic acid (t0)" 2-Methylbutanoic acid (18) Pentanoic acid (28)" Heptanoic acid (33)" Octanoic acid (39)" Nonanoic acid (44)" 6-Methylnonanoic acid (47) Undecanoic acid (53)" 3-Methylbutanoic acid (14)" 4-Methylpentanoic acid (25)" Hexanoie acid (30)" 7-Octenoic acid (36) 2-Methylhexanoic acid (40) 8-Nonenoic acid (45)" Decanoic acid {51 )" Dodecanoic acid (54)" Esters Methyl 2-ethylpropenoate ( 151 Ethyl decanoate (48)" Methyl decanoate (42) Ahlehydes Pentanal (5) Heptanal 120)" Nonanal 134)" Hexanal (8)" OctanaI (47)" K~'IOII~'S
3-Hydroxybulan-2-one ( I 1 Heptan-2-c, ne ( 19)" Nonan-2-one (32)
Ketones Continued Tridecan-7-one (46) 5,Methylhexan-2-one ( 13)" 11-Dodecen-2-one (29) Undecan-2-one (41) Pentadecan-8-one (52) Alcohols Pentan-2-ol (2) Pentan-l-ol (6)" Hexan-2-ol I9)" 3.Methylpentan- I -ol ( 12)" Hexan- I-ol ( 17)" 3-Methylhexan-l-ol (22) Heptan- I-ol I24)" Oclan-1 ol (31)" cis(?l-3-Nonen- I-ol 137) Decan-I-ol (43)" 2-Undecen- I -ol (50) 3-Methylbutan-1-ol t4)" 3-Methy Ibut-2-en-1-ol (7) 4-Methylpentan- 1-ol ( I I I" 5-Methylhexan-2-ol (16) Heptan-2-ol (21 ) cis-3-Hepten- 1-ol (23)" cis(?)-3-Octen- i-oi (26) I-Octen-3-ol (351" Nonan- I-oi (38)" 3-Decen- 1-ol (49)
Su!fi~& Dimethyl disulfide (3t
"Compounds whose identities were confimled by comparison of retention times with those of the authentic compounds,
adult moles. Bacterial activity has been shown to be responsible for volatile carboxylic acids in the anal sacs of the lion (Albone et al., 1974), the red fox (Albone et al., 1974; Ware and Gosden, 1980), and the beaver (Svendsen and Jollick, 1978) and in the inguinal pouch of the rabbit (Merritt et al., 1982), but we have shown (Buglass et al., 1995) that no bacteria are present in the anal gland of the mole, and so carboxylic acids are formed by metabolic processes within the cells of the animal. Bearing in mind that the samples came from different animals, the chromatograms in Figure 2A and B show remarkable similarity in the profile and composition of the volatile components of the anal gland secretion of the mate
390
KHAZANEHDARI, BUGLASS, AND WATERHOUSE
in the nonbreeding and the breeding seasons. The secretions are characterized by relatively high concentrations of carboxylic acids. The secretion from the nonbreeding female (Figure 2D) is also clearly similar in composition to that of the male, showing large carboxylic acid peaks. This corroborates the other malelike characteristics of the nonbreeding female, such as her external genitalia. As the female approaches estrus, however, there are profound changes in the composition of the anal gland secretion (Figure 2E), which occur together with the recession of the glands (Figure I). The carboxylic acids disappear relatively early in the process and, as a consequence, alcohols become the main components for the period during which the glands are still secreting. The pattern in the female secretion is repeated in the rare occurrence of second estrus in September (Figure 2H), but in this case the carboxylic acids had not entirely disappeared at the time the animal was trapped. The characteristic male-like composition of the female secretion is quickly reestablished after estrus, as is shown in the secretion of the pregnant and lactating female (Figure 2F and G, respectively), both of which have significant carboxylic acid peaks. The seasonal changes in the size of the female anal gland and in the composition of its secretion occur concurrently with profound changes in the ovary. The ovary contains two distinct glandular masses: an ovarian part and an interstitial gland, and these features vary in relative size and activity during the year. Outside the breeding season the interstitial gland is far larger than the ovarian part; but during the breeding season the ovarian part enlarges as the interstitial part atrophies, and for a short period the ovarian part is the larger of the two glandular masses (Harrison Matthews, 1935). The enlargement of the ovarian part occurs during the period in which the anal glands regress. After parturition the interstitial gland begins to return to its dominant state as the ovarian part atrophies. The interstitial gland is considered to be homologous to the male testis and to produce male hormones that suppress the norn~al female characteristics outside the breeding season (Harrison Matthews, 1935). The return to dominance of the interstitial tissue after parturition coincides with the reappearance of the male-like composition of the female anal gland secretion. Our results provide further evidence for the role of the anal gland secretion in territorial maintenance, Outside the breeding season, when both sexes deter conspecifics from entering their tunnel systems, the glandular secretions of both sexes are clearly similar in their volatile compositions, which suggests that they produce a common chemical "keep out" signal. For female moles this signal is interrupted only in the breeding season with regression of the anal glands, often to the point of invisibility (Figure 1). It is at this time that a male is able to approach the female and mate. The change in composition of the secretion that accompanies the regression of the gland may produce a chemical signal that alerts a neighboring male mole to the approach of sexual receptiveness in the female, and complete cessation of the secretary activity as regression of the
39 ]
MOLE ANAL GLAND SECRETION
glands is completed may serve to fine tune the mating period; however, a note of caution is required here, for the amount of secretion produced by the regressing glands is very small. It may simply be that males are no longer deterred from entering the female tunnels owing to the switching off o f a keep-out signal; moreover, our results do not preclude the attraction of males by odors from other sources in the receptive female. The rapid reappearance of the male-like composition o f the female secretion after estrus is consistent with the need for the female to protect her tunnel systems during pregnancy, and particularly during lactation, in order to ensure the y o u n g ' s survival. The analytical data suggest that carboxylic acids play a significant semiochemical role. A set o f identical carboxylic acids form the dominant component in the secretion of adult males and anestrous females; it is these carboxylic acids that disappear in the secretion of the proestrous female; they are rapidly reestablished during pregnancy and lactation. The development in the composition of the secretion of juveniles is also instructive. Our analysis showed that the composition of the secretion of both sexes at the same stage o f maturity was very similar and that the composition of the secretion changes as the animal develops and its anal glands increase in size and secretory ability. Figure 2C and 21 illustrate this trend. In the young male caught in May (Figure 2C), carboxylic acids are only very minor components, and the profile is similar to those of the proestrous females (Figures 2E and 2H). Figure 21 shows the chromatogram from a juvenile female caught in late July, This animal was more mature than the juvenile male, although her reproductive tract was still not fully developed. The animal would be nearing the dispersal phase when she would need to set up a territory and mark it as her own. The carboxylic acids in the secretion have now become significant components at this stage in the mole's development. Ackmm'tedgments--We wish to thank David Ellington and lan Smythe of DRE Pest Control. Swavesey, Cambridgeshire, lbr catching and supplying moles, We also thank Anglia Polytechnic University |or financial support.
REFERENCES ALBONE, E.S,, EGLINTON, G., WALKER, J.H., and WARE. G,C. 1974. The anal sac secretion of
the red fox (VMpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of" the lion (Panthera leo). Life Sci. 14:387-400. BUGLASS,A,J., DARLING,F.M.C., and WATERHOUSE,J.S. [990. Analysis of the anal sac secretion of the Hyaenidae, pp. 65-69, in D.W. Macdonald, D. M~iller-Schwarze.and S.E Natynczuk (eds.). Chemical Signals in Vertebrates 5. OUP, Oxford. BUGLASS.A.J., KHAZANEHDAR~.C., and WATERHOUSE.J.S. 1995. Studies on the anal gland of the European mole, pp. 383-387. in R. Apfelbach, D. Miiller-Schwarze, K. Reuter, and E. Weiller (eds.). Chemical Signals in Vertebrates 7. Elsevier, Oxlord.
392
KHAZANEHDARI, BUGLASS, AND WATERHOUSE
CLEVEDONBROWN, J., BUGLASS, A,J., FLOWERDEW,J.R., KHAZANEHDARI,C,, and WATERHOUSE, J.S. 1994. The identity of the enlarged inguinal glands of the mole (Talpa europaea)--anal or preputial? J. Zx~ol, London. 243:674-677. DEANESLY, R. I966. Observations on the reproduction of the mole Talpa europaea. Syrup. ZooL Soc. London t5:387-402. GODFREY,G,, and CROWCROV'T,P. 1960. The Life of the Mole (Talpa europaea Linnaeus). Museum Press, London. GORMAN, M,L., and STONE, R.D. 1989. Repelling moles, pp. 81-97, in R.L Putman (ed.). Mammals as Pests. Chapman and Hall, London. GORMAN, M.L.. and STONE, R.D. 1990. Mutual avoidance by European moles, pp. 367-377, in D.W. Macdonald, D, Mfiller-Schwarze, and S.E~ Natynczuk (eds.). Chemical Signals in Vertebrates 5. OUP, Oxford. HARRISON MATTHEWS, L. 1935. The oestrous cycle and intersexuatity in the female mole. Proc. ZooL Soc. London 347-402. MERmTT, G.C., GOODRICH,B.S., HESTERMAN, E.R., and M','K','TOW'¢CZ, R. 1982. Microflora and volatile fatty acids present in the inguinal pouches of the wild rabbit, Oryctolagus cunictdus in Australia. J. Chem. Ecol. 8:1217-1225. PEARSON, O.P. 1946. Scent glands in the short-tailed shrew. Anat. Rec. 94:6t5-629. RACEr, P.A. 1978. Seasonal changes in testosterone levels and androgen-dependent organs in male moles [Talpa europaea). J. Repro& Fertil. 52: 195-200. STONE, RD., and GORWlAN,M.L. 1985. Social organisation of the European mole (Talpa europaea) and the Pyrenean desman (Galemis py~enaicus). Mammal Rm'. 15:35-42. SVENOSEN, J.E.. and JOLLICK, J.D. 1978. Bacterial contents of the anal and castor glands of the beaver [Castor canadensis). J. Chem. Ecol. 4:563-569. WARE, G,C., and GOSDEN, P.E. 1980, Anaerobic microflora in the anal sac of the red fox (Vulpes vulpes). J. Chem. Ecol. 6:97-102.
ANAL GLAND SECRETION OF EUROPEAN MOLE: VOLATILE CONSTITUENTS AND SIGNIFICANCE IN TERRITORIAL MAINTENANCE
CHANTAL
KHAZANEHDARI, and JOHN
ALAN
J. B U G L A S S ,
S. W A T E R H O U S E *
Enlqronmental Science Research Centre Anglia Polytechnic University East Rd., Cambridge. CBt IPT. U.K~ (Received June 6, 1995; accepted October 16, 1995)
Abstract--The chemical composition of the volatile components of the anal gland secretion of mature and immature moles of both sexes was examined by gas chromatography and mass spectrometry. The compounds in the secrelion of adult males appear to vary little throughout the year and are similar to those from the adutt female outside the breeding season. The secretions are dominated by C5-C~o carboxylic acids. Female glands regress as they enter proestms, accompanied by profound changes in the chemical composition of the secretion with early disappearance of the carboxylic acids. In juvenile moles, the compostion of the secretion changes as the animal matures, with carboxylic acids becoming dominant only as the animal reaches maturity. Anal gland secretion probably plays an important role in territorial maintenance. Key Words--Moles, Talpa europaea, anal glands, scent marking, territoriality, semiochemical, proestrus, carboxylic acids.
INTRODUCTION F o r m o s t o f the y e a r t h e E u r o p e a n m o l e (Talpa europaea) l i v e s a s o l i t a r y life in its o w n s u b t e r r a n e a n t u n n e l s y s t e m a n d a c t i v e l y a v o i d s c o n t a c t w i t h o t h e r m e m b e r s o f its o w n s p e c i e s . T h e h o m e r a n g e s o f f e m a l e s o r o f a m a l e a n d a f e m a l e m a y , h o w e v e r , o v e r l a p , a n d l e n g t h s o f t u n n e l m a y be u s e d in c o m m o n , M o l e s in o v e r l a p p i n g t e r r i t o r i e s are a w a r e o f e a c h o t h e r ' s e x i s t e n c e a n d m o v e *To whom correspondence should be addressed. 383 00984)33t/96/02fX~-0383~)9511/0~c~1996PlcnumPublishingCorpl~r,~li~n
384
KI-IAZANF~HI)ARI. BI.~(;LASS. AND WAFERI-IOIIN~:
ments, and they will organize their daily routine so as to avoid each other (Stone and Gorman, 1985; Gorman and Stone, 1990), The only time of the year when moles purposefully meet is during the mating season, when male moles will migrate into female territories in order to mate (Godfrey and Crowcroft, 1960). Studies by Gorman and Stone (1990) have suggested that the mole's efficient territorial maintenance is controlled at least in part by chemical signals produced by the secretion of specialized glands in the inguinal region of both sexes. These glands have been termed "'preputial glands" by some workers (Racey, 1978; Stone and Gom~an, 1985; Gorman and Stone, 1989, 1990), but we have recently shown that they are in fact anal glands (Clevedon Brown et al., 1994). We present results here that support the hypothesis that the secretion from the anal glands plays a central role in the territorial maintenance of the mole. Our evidence is based on studies on variations in glandular mass and the chemical composition of the volatile components of the glandular secretion throughout the year. METHODS AND MATERIALS
Moles used for both the anatomical and chemical studies were caught in lethal traps in Cambridgeshire and adjacent parts of Norfolk by a professional mole catcher (DRE Pest Control, Swavesey, Cambridgeshire). The carcasses were frozen immediately on collection for transport to the laboratory, where they remained frozen at - 2 0 ° C until dissection. Anal glands were separated from extraglandular material and weighed. The glandular secretion was released by crushing the gland, and the volatile components therein were trapped on an activated charcoal disc using a Gorb dynamic headspace apparatus as previously described (Buglass et al., 1990). The volatiles were eluted from the disc with 8 #1 of dichloromethane containing dihexyl sulfide (115 ng/txl as internal standard), and 1 p.l of the resulting solution injected into a Hewlett-Packard 5890 gas chromatograph equipped with a polydimethylsiloxane column (length, 15 m; ID, 0,25 ram) and connected to a Hewlett-Packard 5970 mass spectrometer detector. The temperature program was 35-240°C at a rate of 4°C/rain with a final hold at 240°C for 45 min. Compounds were identified from their mass spectra and also, in most cases, by comparison of retention times with those of authentic compounds (see Table 1 below). Male moles in breeding condition were distinguished from nonbreeding males by the great increase in size of the testes and epidydimus. Proestrus in the female was characterized by the presence of the vaginal opening, the increase in length and breadth of the reproductive tract resulting from growth and thickening of its wall, and vascularization of the uterine cornua (Hamson Matthews, 1935). We observed an increase in length of the uterovaginal canal to around
MOLE ANAL GLAND SECRETION
385
40-45 % of body length during proestrus, as opposed to a normal value of less than 20%, in line with the findings of Harrison Matthews (1935). As estrus itself lasts for only about 30 hr in the mote (Godfrey and Crowcroft, 1960L no attempt was made to distinguish between the proestrous and the estrous female.
RESULTS A N D D I S C U S S I O N
In East Anglia the mating season for moles occurs early in the year. Females approach estrus as early as January. The gestation period is four weeks and lactation follows tot a further four weeks (Deanesly, 1966: Godfrey and Crowcroft, 1960: Harrison Matthews, 1935): hence by February or March, females are likely to be pregnant or to be lactating. Additionally, we have observed a low incidence of second proestrus and presumably estrus in September. Preliminary results from our study of the changes in anal gland size during the year showed that the glands of males and females behave differently (Buglass et al., 1995). Male glands increase in weight during the breeding season, reaching a maximum value at the peak of the breeding season in February and March. This change is probably due to vascular development resulting from a surge in testosterone, which in the shrew serves to increase vascularity of sebaceous tissue (Pearson, 1946). The increase in size and weight of the gland is associated with a deepening of its color to dark red. In contrast, female glands undergo a reduction in size as the female enters estrus in January or February. In the majority of 14 females that were collected in January. the glands had regressed to such a degree that they were invisible to the naked eye. Female glands enlarge as the breeding period ends, but the average value drops sharply in May before climbing again during summer and fall. We explain this effect by the inclusion of juveniles in the catch tYom May onwards. The glands of juveniles are flaccid and less developed than those of the mature mole, and it is not until August or September that glands of moles born in that year become indistinguishable from those of mature animals. The results of an extension of this study, involving a greater number of moles, are shown in Figure 1. These results are in full agreement with our earlier findings. Samples of anal gland secretion from a total of 130 moles were analyzed by gas chromatography and mass spectrometry. For adult moles, chromatograms were obtained from secretion collection in each month of the year. A typical chromatogram from a nonbreeding male, a breeding male, a juvenile male, a nonbreeding female, a proestrous female, a pregnant female, a lactating female, a female approaching second estrus, and a juvenile female are shown in Figure 2A-I, respectively, Compounds are identified in Table 1. With the exception of the proestrous female, C s-Cto carboxytic acids, both straight and branched chain, tbrm a significant component of the secretions of
386
KHAZANEHDARI, BUGLASS, AND WA]ERH()USE
1
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0.3
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FiG. t. Box and whisker plots showing the seasonal changes in the anal gland weight as a percentage of body weight for male and female moles. Numbers refer to number of moles in each sample.
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387
SECRETION
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FIG. 2, Gas chromatograms of volatile compounds from the anal gland secretions of moles: (A) nonbreeding male (October); (B) breeding male (April): (C) juvenile male (May); (D) nonbreeding female (October): (E) proestrous female (January); (F) pregnant female (February); (G) lactating female (March); (H) female approaching second estrus (September): (I) juvenile female (May). x = impurity; IS = internal standard (dihexyl sulfide).
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389
MOLE ANAL GLAND SECRETION TABLE [. COMPOUNDS IDENTIFIED IN ANAL GLAND SECRETION OF MOLE ( N u m b e r s in parentheses match those in Fig. 2.)
Carbo.~,lic acids Butanoic acid (t0)" 2-Methylbutanoic acid (18) Pentanoic acid (28)" Heptanoic acid (33)" Octanoic acid (39)" Nonanoic acid (44)" 6-Methylnonanoic acid (47) Undecanoic acid (53)" 3-Methylbutanoic acid (14)" 4-Methylpentanoic acid (25)" Hexanoie acid (30)" 7-Octenoic acid (36) 2-Methylhexanoic acid (40) 8-Nonenoic acid (45)" Decanoic acid {51 )" Dodecanoic acid (54)" Esters Methyl 2-ethylpropenoate ( 151 Ethyl decanoate (48)" Methyl decanoate (42) Ahlehydes Pentanal (5) Heptanal 120)" Nonanal 134)" Hexanal (8)" OctanaI (47)" K~'IOII~'S
3-Hydroxybulan-2-one ( I 1 Heptan-2-c, ne ( 19)" Nonan-2-one (32)
Ketones Continued Tridecan-7-one (46) 5,Methylhexan-2-one ( 13)" 11-Dodecen-2-one (29) Undecan-2-one (41) Pentadecan-8-one (52) Alcohols Pentan-2-ol (2) Pentan-l-ol (6)" Hexan-2-ol I9)" 3.Methylpentan- I -ol ( 12)" Hexan- I-ol ( 17)" 3-Methylhexan-l-ol (22) Heptan- I-ol I24)" Oclan-1 ol (31)" cis(?l-3-Nonen- I-ol 137) Decan-I-ol (43)" 2-Undecen- I -ol (50) 3-Methylbutan-1-ol t4)" 3-Methy Ibut-2-en-1-ol (7) 4-Methylpentan- 1-ol ( I I I" 5-Methylhexan-2-ol (16) Heptan-2-ol (21 ) cis-3-Hepten- 1-ol (23)" cis(?)-3-Octen- i-oi (26) I-Octen-3-ol (351" Nonan- I-oi (38)" 3-Decen- 1-ol (49)
Su!fi~& Dimethyl disulfide (3t
"Compounds whose identities were confimled by comparison of retention times with those of the authentic compounds,
adult moles. Bacterial activity has been shown to be responsible for volatile carboxylic acids in the anal sacs of the lion (Albone et al., 1974), the red fox (Albone et al., 1974; Ware and Gosden, 1980), and the beaver (Svendsen and Jollick, 1978) and in the inguinal pouch of the rabbit (Merritt et al., 1982), but we have shown (Buglass et al., 1995) that no bacteria are present in the anal gland of the mole, and so carboxylic acids are formed by metabolic processes within the cells of the animal. Bearing in mind that the samples came from different animals, the chromatograms in Figure 2A and B show remarkable similarity in the profile and composition of the volatile components of the anal gland secretion of the mate
390
KHAZANEHDARI, BUGLASS, AND WATERHOUSE
in the nonbreeding and the breeding seasons. The secretions are characterized by relatively high concentrations of carboxylic acids. The secretion from the nonbreeding female (Figure 2D) is also clearly similar in composition to that of the male, showing large carboxylic acid peaks. This corroborates the other malelike characteristics of the nonbreeding female, such as her external genitalia. As the female approaches estrus, however, there are profound changes in the composition of the anal gland secretion (Figure 2E), which occur together with the recession of the glands (Figure I). The carboxylic acids disappear relatively early in the process and, as a consequence, alcohols become the main components for the period during which the glands are still secreting. The pattern in the female secretion is repeated in the rare occurrence of second estrus in September (Figure 2H), but in this case the carboxylic acids had not entirely disappeared at the time the animal was trapped. The characteristic male-like composition of the female secretion is quickly reestablished after estrus, as is shown in the secretion of the pregnant and lactating female (Figure 2F and G, respectively), both of which have significant carboxylic acid peaks. The seasonal changes in the size of the female anal gland and in the composition of its secretion occur concurrently with profound changes in the ovary. The ovary contains two distinct glandular masses: an ovarian part and an interstitial gland, and these features vary in relative size and activity during the year. Outside the breeding season the interstitial gland is far larger than the ovarian part; but during the breeding season the ovarian part enlarges as the interstitial part atrophies, and for a short period the ovarian part is the larger of the two glandular masses (Harrison Matthews, 1935). The enlargement of the ovarian part occurs during the period in which the anal glands regress. After parturition the interstitial gland begins to return to its dominant state as the ovarian part atrophies. The interstitial gland is considered to be homologous to the male testis and to produce male hormones that suppress the norn~al female characteristics outside the breeding season (Harrison Matthews, 1935). The return to dominance of the interstitial tissue after parturition coincides with the reappearance of the male-like composition of the female anal gland secretion. Our results provide further evidence for the role of the anal gland secretion in territorial maintenance, Outside the breeding season, when both sexes deter conspecifics from entering their tunnel systems, the glandular secretions of both sexes are clearly similar in their volatile compositions, which suggests that they produce a common chemical "keep out" signal. For female moles this signal is interrupted only in the breeding season with regression of the anal glands, often to the point of invisibility (Figure 1). It is at this time that a male is able to approach the female and mate. The change in composition of the secretion that accompanies the regression of the gland may produce a chemical signal that alerts a neighboring male mole to the approach of sexual receptiveness in the female, and complete cessation of the secretary activity as regression of the
39 ]
MOLE ANAL GLAND SECRETION
glands is completed may serve to fine tune the mating period; however, a note of caution is required here, for the amount of secretion produced by the regressing glands is very small. It may simply be that males are no longer deterred from entering the female tunnels owing to the switching off o f a keep-out signal; moreover, our results do not preclude the attraction of males by odors from other sources in the receptive female. The rapid reappearance of the male-like composition o f the female secretion after estrus is consistent with the need for the female to protect her tunnel systems during pregnancy, and particularly during lactation, in order to ensure the y o u n g ' s survival. The analytical data suggest that carboxylic acids play a significant semiochemical role. A set o f identical carboxylic acids form the dominant component in the secretion of adult males and anestrous females; it is these carboxylic acids that disappear in the secretion of the proestrous female; they are rapidly reestablished during pregnancy and lactation. The development in the composition of the secretion of juveniles is also instructive. Our analysis showed that the composition of the secretion of both sexes at the same stage o f maturity was very similar and that the composition of the secretion changes as the animal develops and its anal glands increase in size and secretory ability. Figure 2C and 21 illustrate this trend. In the young male caught in May (Figure 2C), carboxylic acids are only very minor components, and the profile is similar to those of the proestrous females (Figures 2E and 2H). Figure 21 shows the chromatogram from a juvenile female caught in late July, This animal was more mature than the juvenile male, although her reproductive tract was still not fully developed. The animal would be nearing the dispersal phase when she would need to set up a territory and mark it as her own. The carboxylic acids in the secretion have now become significant components at this stage in the mole's development. Ackmm'tedgments--We wish to thank David Ellington and lan Smythe of DRE Pest Control. Swavesey, Cambridgeshire, lbr catching and supplying moles, We also thank Anglia Polytechnic University |or financial support.
REFERENCES ALBONE, E.S,, EGLINTON, G., WALKER, J.H., and WARE. G,C. 1974. The anal sac secretion of
the red fox (VMpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of" the lion (Panthera leo). Life Sci. 14:387-400. BUGLASS,A,J., DARLING,F.M.C., and WATERHOUSE,J.S. [990. Analysis of the anal sac secretion of the Hyaenidae, pp. 65-69, in D.W. Macdonald, D. M~iller-Schwarze.and S.E Natynczuk (eds.). Chemical Signals in Vertebrates 5. OUP, Oxford. BUGLASS.A.J., KHAZANEHDAR~.C., and WATERHOUSE.J.S. 1995. Studies on the anal gland of the European mole, pp. 383-387. in R. Apfelbach, D. Miiller-Schwarze, K. Reuter, and E. Weiller (eds.). Chemical Signals in Vertebrates 7. Elsevier, Oxlord.
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KHAZANEHDARI, BUGLASS, AND WATERHOUSE
CLEVEDONBROWN, J., BUGLASS, A,J., FLOWERDEW,J.R., KHAZANEHDARI,C,, and WATERHOUSE, J.S. 1994. The identity of the enlarged inguinal glands of the mole (Talpa europaea)--anal or preputial? J. Zx~ol, London. 243:674-677. DEANESLY, R. I966. Observations on the reproduction of the mole Talpa europaea. Syrup. ZooL Soc. London t5:387-402. GODFREY,G,, and CROWCROV'T,P. 1960. The Life of the Mole (Talpa europaea Linnaeus). Museum Press, London. GORMAN, M,L., and STONE, R.D. 1989. Repelling moles, pp. 81-97, in R.L Putman (ed.). Mammals as Pests. Chapman and Hall, London. GORMAN, M.L.. and STONE, R.D. 1990. Mutual avoidance by European moles, pp. 367-377, in D.W. Macdonald, D, Mfiller-Schwarze, and S.E~ Natynczuk (eds.). Chemical Signals in Vertebrates 5. OUP, Oxford. HARRISON MATTHEWS, L. 1935. The oestrous cycle and intersexuatity in the female mole. Proc. ZooL Soc. London 347-402. MERmTT, G.C., GOODRICH,B.S., HESTERMAN, E.R., and M','K','TOW'¢CZ, R. 1982. Microflora and volatile fatty acids present in the inguinal pouches of the wild rabbit, Oryctolagus cunictdus in Australia. J. Chem. Ecol. 8:1217-1225. PEARSON, O.P. 1946. Scent glands in the short-tailed shrew. Anat. Rec. 94:6t5-629. RACEr, P.A. 1978. Seasonal changes in testosterone levels and androgen-dependent organs in male moles [Talpa europaea). J. Repro& Fertil. 52: 195-200. STONE, RD., and GORWlAN,M.L. 1985. Social organisation of the European mole (Talpa europaea) and the Pyrenean desman (Galemis py~enaicus). Mammal Rm'. 15:35-42. SVENOSEN, J.E.. and JOLLICK, J.D. 1978. Bacterial contents of the anal and castor glands of the beaver [Castor canadensis). J. Chem. Ecol. 4:563-569. WARE, G,C., and GOSDEN, P.E. 1980, Anaerobic microflora in the anal sac of the red fox (Vulpes vulpes). J. Chem. Ecol. 6:97-102.
