Chem. Senses 28: 433-446,
2003
© Oxford University Press 2003
Frontalin: a Chemical Message of Musth in Asian Elephants (Elephas maximus)
1 Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, OR 97006-8921, USA 2 The Horticulture and Food Research Institute of New Zealand Limited, Private Bag 92-169, Auckland, New Zealand
Correspondence to be sent to: L.E.L. Rasmussen, Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, 20000 N.W. Walker Road, Beaverton, OR 97006-8921, USA. e-mail: betsr{at}bmb.ogi.edu
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Abstract |
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Musth is an important male phenomenon affecting many aspects of elephant society including reproduction. During musth, the temporal gland secretions (as well as the urine and breath) of adult male Asian elephants (Elephas maximus) discharge a variety of malodorous compounds together with the bicyclic ketal, frontalin. In contrast, teenage male elephants in musth release a sweet-smelling exudate from their facial temporal gland. We recently demonstrated that the concentration of frontalin becomes increasingly evident as male elephants mature. In the present study, we demonstrate that behaviors exhibited towards frontalin are consistent and dependent on the sex, developmental stage and physiological status of the responding conspecific individual. To examine whether frontalin functions as a chemical signal, perhaps even a pheromone, we bioassayed older and younger adult males, and luteal- and follicular-phase and pregnant females for their chemosensory and behavioral responses to frontalin. Adult males were mostly indifferent to frontalin, whereas subadult males were highly reactive, often exhibiting repulsion or avoidance. Female chemosensory responses to frontalin varied with hormonal state. Females in the luteal phase demonstrated low frequencies of responses, whereas pregnant females responded significantly more frequently, with varied types of responses including those to the palatal pits. Females in the follicular phase were the most responsive and often demonstrated mating-related behaviors subsequent to high chemosensory responses to frontalin. Our evidence strongly suggests that frontalin, a well-studied pheromone in insects, also functions as a pheromone in the Asian elephant: it exhibits all of the determinants that define a pheromone and evidently conveys some of the messages underlying the phenomenon of musth.
Key words: chemical senses, 1,5-dimethyl-6, 8-dioxabicyclo[3.2.1]octane, female elephant behavior, male elephant behavior, pheromone, temporal gland
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Asian elephants (Elephas maximus) rely heavily on their chemical senses to make decisions about foraging and migration, to recognize genetic and social relationships, to identify and choose mates, and to establish and maintain social order (Rasmussen and Krishnamurthy, 2000
). Male elephants are an intimate part of the
family group as youngsters, but they become increasingly adventuresome
(Sukumar, 1989), foraging for
a greater variety of fodder farther from the family unit
(McKay, 1973). Males mature
sexually, from an anatomical perspective, as teenagers
(Hildebrandt et al.,
2000) but do not mature socially until their twenties, concurrent
with regular musth episodes (Jainudeen et al.,
1972a,b).
As teenagers or before, young males have sporadic, short musth episodes
chemically characterized by facial temporal gland emissions of sweet-smelling
acetates, alcohols, and ketones, elevated oscillating levels of serum
androgens, and periods of erratic behavior
(Rasmussen et al.,
2002). However, as fully mature adult males, their musth periods
lengthen, serum androgen and triglycerides elevate greatly, and their
behaviors become overtly aggressive
(Rasmussen and Perrin, 1999).
Their desire for physically appropriate mates increases. Concomitantly, the
chemical composition of the temporal gland secretions (TGS) changes
dramatically (Rasmussen et al.,
2002).
Male behavior during musth is influenced by internal physiological changes.
In turn, the musth behavior affects the physiology and behavior of
conspecifics, having ultimate and proximate influences on reproduction.
Ultimately, the musth condition may release `honest' chemical signals and
influence the reproductive behavior of females, perhaps via the handicap
principle as suggested by Nath (Nath,
1999). For males, recognition of the ontogenic degree of musth
prior to physical encounters is clearly advantageous. Proximately, the musth
condition may influence male behavior or may induce reactions by conspecifics,
either male or female. For males, immediate reactions are maturity and musth
state-related, and include male–male spacing, overt avoidance, or
attraction with accompanying flehmen responses to exudates such as urine or
temporal gland secretions (Rasmussen
et al., 2002). For example, from a proximate perspective,
observations in the wild, coupled with instantaneous chemical sampling and
captive elephant playback experiments, demonstrated that younger, socially
immature males in musth may signal their naïveté by releasing
honey-like odors to avoid conflicts with adult males, whereas older musth
males broadcast malodorous combinations to deter young males
(Rasmussen et al.,
2002). These immediate and often lasting effects facilitate the
smooth functioning of male society.
A similar duet of proximate and ultimate exchanges of chemical signals
resulting in altered or informed behaviors between musth males and females may
also be operational. During controlled playback studies, some female elephants
avoided and retreated from many, but not all, fresh mature male musth TGS
samples, whereas only a few freezer-stored (–20°C) samples were
effective (Perrin et al.,
1996). Since only subordinate females exhibited the retreat
behaviors, the tested population was too small to reach a meaningful
conclusion, although dominance appeared to be a factor. Concurrent chemical
analyses demonstrated that a specific complement of ketones and alcohols and a
bicyclic ketal compound were always present in the samples that elicited
bioresponses (Perrin et al.,
1996).
Since the ketonic compounds elicited variable behavioral responses, we
focused our attention on the bicyclic ketal, frontalin
(1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane)
(Figure 1), a
well-characterized bark beetle pheromone
(Kinzer et al., 1969;
Phillips et al.,
1990; Lindgren,
1992). In the Asian elephant, frontalin is present in the TGS,
urine, and breath of mature males in musth
(Rasmussen, 1998). However,
frontalin is absent in the TGS of subadult male elephants experiencing their
first or moda musths, only becoming detectable in the TGS as the males mature
and experience successive, more adult-like musth episodes
(Rasmussen et al.,
2002). Bioassays of whole TGS with male elephants and observations
in the wild of male–male behavior demonstrated that older males are
somewhat indifferent to moda musth males and their secretions (which lack
frontalin), while younger adolescent males avoid TGS and older males in musth
(Rasmussen et al.,
2002). Thus, there is a congruence of chemical and behavioral
events evident in male–male interactions.
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Initial tests also suggested that female elephants respond differently to TGS containing or lacking frontalin (L.E.L. Rasmussen, unpublished data). The documentation of distinctive chemosensory responses and behaviors toward frontalin by males and females would elucidate whether frontalin plays a role as a chemical signal in reproduction. Such information should allow elephants to recognize male musth state prior to physical encounters and to adjust behavioral responses appropriately. Therefore, we hypothesized that Asian elephants recognize this male-emitted compound chemosensorily, employing it to assess the sexual maturity and/or musth state of male conspecifics, and that this recognition would be evident in behavioral reactions and responses that maintain social order and hierarchy. To test our hypotheses, we designed a series of bioassays to assess the behavioral and chemosensory responses of elephants of both sexes to synthetic frontalin. We present the results and interpretation of these experiments, analyzed from the perspective of the sex, maturity, and hormonal status of the tested elephants that strongly implicate frontalin as having a distinct chemical messaging functionality in Asian elephants.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Elephants and facilities
A total of 30 elephants, 21 females and nine males, were available for this study. The elephants were housed at Riddle's Elephant Sanctuary (RES), Greenbrier, AR (n = 4), the Ringling Center for Elephant Conservation (RCEC), Polk City, FL (n = 23), and the Oregon Zoo, Portland, OR (n = 3). Each facility had a sizable enclosure yard available for bioassays.
All bioassays with male elephants were conducted on nonmusth animals.
Nonmusth was defined by behavioral characteristics and physiological
parameters including serum testosterone concentrations below 10 ng/ml
(Rasmussen and Perrin, 1999).
Among males, two categories of sexual maturity were recognized: (i) subadults,
i.e. sexually mature but socially immature teenage or sub-teenage males,
n = 3, and (ii) older adults, i.e. fully socially and sexually mature
males, ranging in age from 27 to 34 years, n = 6.
The female elephants were all sexually mature. All were cycling except four pregnant elephants. The hormonal status of the 17 cycling females was ascertained after the bioassays were completed. Serum concentrations of the steroid hormone, progesterone, were then used to categorize the females prior to data analysis.
Hormonal data
Serum hormone concentrations (testosterone in males and progesterone in
females) were measured by radioimmunoassay by Dr David Hess (Oregon Health
& Science University, Beaverton, OR), Dr Thomas Goodwin (Hendrix College,
Conway, AR), and by Ringling Center for Elephant Conservation (Polk City, FL).
Sensitivity for testosterone and progesterone levels was 10 and 15 pg/tube,
respectively, at 90% binding levels using validated assays (Resko et
al., 1973,
1980;
Hess et al., 1983;
Rasmussen et al.,
1984).
Bioassays
The bioassay scheme has been detailed previously (Rasmussen et
al., 1982,
1986,
1996). The present
investigation was conducted as a double-blind study, i.e. the investigator was
not aware of the identity of the samples, and the elephant did not see the
samples being placed. Two samples were randomly placed on ground-level
concrete areas, at least 20 feet apart, prior to the entry of the test
elephant(s). Temperature, moisture, and wind speed and direction were
recorded. Elephants were observed for 1 h from a distance adequate to see
clearly but non-distracting to the animal. All occurrences of standard
chemosensory responses and evoked behaviors described below were recorded for
the focal animal(s) using simultaneous voice and video recording. All assays
with males were conducted on solitary individuals. Individual males were
repetitively tested, a total of six times.
All females were assayed as part of a group, not as solitary subjects. Between one and 10 tests, each separated by 3–6 month intervals, were conducted on 21 female elephants over a period of 5 years. Females were either assayed one time only in their luteal, follicular or pregnant stages, or they were assayed repetitively, for a total of five times each in the luteal and follicular phases or three times each while pregnant. Except for the pregnant elephants, the estrous status was unknown to the investigator at the time of the assay. Since the estrous cyclicity varied among females, single assays of females in either their luteal or follicular phase were easily obtained. The testing of multiple-assayed females was randomized between the luteal and follicular phases by varying the number of months between bioassays. After the assays, the data from the cycling females were sorted into two reproductive categories, based on serum progesterone concentrations: follicular (F) or luteal (L).
Responses and behaviors
Scored olfactory-implicated responses included sniffs, trunk shakes, blows
(forceful exhalations), checks, places, sucks and flehmens. The data were
expressed as responses per hour. During data analyses the responses were
categorized as main olfaction (sniffs, trunk shakes and blows), preflehmen
(checks, places and sucks) and flehmen. Sniffs by Asian elephants are clearly
delineated by overt directionality of the trunk tip, slight compression of
trunk musculature, and sometimes-audible inhalation, with involvement of the
main olfactory system being assumed. Trunk shakes are mild exhalations that
occur subsequent to sniffs and some pre-flehmen responses. Blows are forceful
exhalations of air through the trunk. Both of these latter responses are
presumably for the purpose of clearing odorants from the truncal passages. In
the pre-flehmen response, the check, only the trunk tip finger is placed in
the sample area; slight trunk compression is often noticed and inhalation may
be heard. The place response involves the placement of the entire trunk tip
flush with the sample area on the ground. During a suck, the entire trunk tip
is also flush with the substrate and there is accompanying trunk compression,
often with audible inhalation. The main olfactory system is postulated to be
at least partially involved in the three pre-flehmen responses
(Rasmussen and Schulte, 1998).
During the flehmen response, the trunk tip finger containing drops of mucus
accompanied often with urine or sample is placed precisely on the openings of
the vomeronasal duct on the dorsal anterior palate; the vomeronasal organ is
implicated in this response (Rasmussen
et al., 1982; Lazar
et al., 2002).
The recorded evoked behaviors included avoidance (circling motions around samples with no contact), repulsion (backing up or moving away from the sample after chemosensory responses such as main olfactory, preflehmen or flehmen responses), vocalizations (trumpeting and roaring), and conspecific palatal pit responses. Palatal pit responses are characterized by an elephant placing its trunk tip into the palatal pits, small hair-filled crypts located in the oral mucosa lateral to the vomeronasal organ openings (A. Hansen and L.E.L. Rasmussen, unpublished data). Placement may be to the elephant's own palatal pits or to those of a conspecific. Sender and receiver elephants were identified for the latter responses. Indifference to samples was also recorded. The sequences of chemosensory responses and evoked behaviors were recorded as the total number of occurrences during the bioassay hour.
Samples
In each assay, paired samples were presented: 100 µM frontalin in 0.01 M
phosphate buffer, pH 8.0, and buffer control. (+/-)-Frontalin was purchased as
a racemate from Pherotech (Delta, BC, Canada) and used without further
purification. We estimated the physiologically reasonable test concentration
(100 µM) based on levels of frontalin measured in natural temporal gland
volatiles (Perrin et al.,
1996; Rasmussen et
al., 2002).
When studying captive elephants, there are logistical challenges and
unforeseen variables; experimental conditions are sometimes less than ideal
compared with small mammals. The cognitive, olfactory-oriented elephant is
curious about novel odors and mixtures. Extensive experience in elephant
testing has demonstrated the necessity for repetitive testing to sort out
responses to novel mixtures from responses to biologically meaningful samples
(Rasmussen et al.,
1986). With valid chemical signals or pheromones, responses do not
diminish over time in mature elephants of constant hormonal status, although
some variation between individual elephants may be observed. We are aware of
the problem of pseudoreplication. Because of the novel substance response
phenomenon, we deliberately tested selected elephants, both male and female,
six and five times, respectively, over several years to include the females'
luteal and follicular estrous states. The data were analyzed from several
perspectives and groupings. In Tables
1,
4,
8 and
9, the analyses from the first
bioassays are presented. In Tables
2,
5 and
10, repetitive test data are
presented. We present ranges and averages so that attributes of the individual
assays are more evident. Tables
3 and
7 detail the behavioral
sequences.
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Data analysis
Male elephants were categorized by maturity status and age, whereas female elephants were grouped according to reproductive status: luteal phase, follicular phase or pregnant. Because part of this study involved repeated measures of the same individual males and females, these measurements are not independent and tests such as the Mann–Whitney Rank Sum are only applicable when first assays are compared. In addition, the data included zeros, which presents another issue, necessitating statistical restraint. For the males, we tested three subadult males six times, and six adult males six times. Initially, only the first assay results of the three subadult and the six older males were analyzed by the Mann–Whitney Rank Sum test (Table 1). Subsequently, the response frequencies were compared for repetitively tested younger and older males (Table 2). The data are listed as ranges, averages, and medians; repeated measures of one-way analysis of variance (ANOVA) and pair-wise comparisons with the Student–Newman–Keuls test were conducted (Tables 1 and 2).
The study of the 21 female elephants included those in three hormonal phases: follicular, luteal, and pregnant. We hypothesized that females in the follicular phase would be more responsive to the musth state of the male as revealed by frontalin. We assayed six female elephants in the luteal phase one time only and six different females in the follicular phase also one time only. The data were tabulated and statistically compared using the Mann–Whitney Rank Sum test (Table 4).
We wanted to confirm, by repetitive testing, the data from the first tests.
We hypothesized that if frontalin was a valid reproductive chemical signal
(i.e. a pheromone), operational from male Asian elephants to conspecific
females, then females would also respond more during their follicular rather
than their luteal phase. Balanced data sets were obtained for five different
female elephants, repetitively tested five times each in their follicular and
luteal phases (Tables 5 and
6). We tested this confirmatory
hypothesis through the use of repeated measures of ANOVA using Statistica
(StatSoft, Inc.). Since we had the required balanced data sets, repeated
measures analysis avoided some pseudoreplication issues
(Schulte and Rasmussen, 1999).
During ANOVA analyses of the repetitive female data, the phase in estrus was
the independent variable while the sample type (frontalin versus buffer) and
the tests (1–6) were the repeated measures. We only statistically
compared main olfactory and pre-flehmen responses between luteal- and
follicular-phase females (Table
7), because luteal-phase females exhibited no flehmen responses
(Table 6). We report the data
for flehmen responses, but because of the zeros in the flehmen data, we
focused on the main olfactory and pre-flehmen responses.
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We postulated that pregnant elephants, perhaps in critical stages of pregnancy, need to be aware of the state of the male. Our data from pregnant elephants are less comprehensive than for cycling females. Testing was somewhat more opportunistic and more variables were present. For example, assays were conducted at varying times during the 22 month gestation period. As a first test, we were able to assay four pregnant elephants in varying stages of pregnancy (Tables 8 and 9). Three elephants, including two that were assayed first during their non-pregnant state, were repetitively assayed three times in the pregnant state (Table 10).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The responses by male and female elephants to the buffer only were limited to an occasional sniff or check when presented paired with 100 µM frontalin buffer in each assay. Therefore only the data from responses to frontalin are presented.
Males
Individual males were tested six times with placed samples of 100 µM frontalin, paired to buffer controls. The response data were analyzed in terms of first tests and all tests (Table 1). The first assays with the younger males demonstrated high and tightly distributed chemosensory responses, including main olfactory (MO) responses, pre-flehmen responses and flehmens (Table 1). In contrast, male Asian elephants in the fully mature category (ages 26–38 years) responded at very low levels to frontalin. Except for one older, subdominant male (OA1), older males only responded with a few sniffs and checks that were randomly scattered among the assays. The data from the first bioassays with the younger subadult males were compared with those of the older males and analyzed by Mann–Whitney Rank Sum tests. Statistical tests demonstrated that the three subadult males (SA1–SA3) performed significantly greater numbers of pre-flehmen and flehmen responses to frontalin than the six older males (OA1–OA6) (Table 1), whereas no significant differences were observed in the main olfaction (MO) categorized responses (Table 1).
Repetitive testing generally confirmed the results seen in the first tests, i.e. that the subadult males performed significantly more flehmen and pre-flehmen responses to frontalin than the older males, but there was little difference in MO responses between the two groups (Table 2). However, as repetitive testing allowed more extensive comparison of individual males, some individual differences were revealed. Older males rarely exhibited flehmen; the exception was older male OA1 whose flehmen response frequency was not statistically different from SA2 and SA3. Also, subadult male SA1 demonstrated significantly greater flehmen responses than all six older males and SA2 and SA3 (Table 2). All older adults, except OA1, showed significantly reduced pre-flehmen responses compared to those of all subadult males but with varying degrees of significance (Table 2). Older male OA1 performed pre-flehmen responses that were significantly different from subadult male SA1 and SA2, but not SA3. One older male OA4 was sufficiently non-responsive during repetitive testing that its olfactory categorized responses were significantly lower than those of all subadult males. Figure 2 summarizes the response differences between subadult and older males as well as between groups of females.
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Sequential chemosensory–behavioral responses by male elephants after sampling the placed frontalin samples were sorted into three categories: attraction, indifference, and repulsion/avoidance (Table 3). Three younger males (ages 7–13 years), tested over several years, demonstrated strong reactions of backing up (repulsion) and avoidance (circling around) to frontalin samples with accompanying roaring and trumpeting vocalizations. The older males exhibited indifference in correlation with chemosensory responses and exhibited no vocalizations or aversive movements (Table 3).
Females
Serum progesterone (P4) concentrations for the only-once-tested
females ranged between undetectable and 92 pg/ml for the six follicular-phase
females (A, D, H, I, M and O), and between 340 and 1294 pg/ml for the six
different luteal-phase females (C, E, F, N, U and Y)
(Table 4). Five of the 21
females (B, J, P, S and T) were repeatedly tested in five assays during the
follicular phase, and the same five females were repeatedly tested five times
during the luteal phase (Tables
5 and
6). Their P4
concentrations were in the ranges 6–62 pg/ml and 404–1069 pg/ml,
respectively. Repetitive testing demonstrated that responses to frontalin were
robust. There was no evidence of novel substance response
(Rasmussen et al.,
1986).
First tests of six follicular-phase females and six different luteal-phase females demonstrated that MO and pre-flehmen responses by follicular-phase females were significantly higher than those by luteal-phase females as seen by averages, ranges and medians, and confirmed by Mann–Whitney Rank Sum test (Table 4). Flehmens were not significantly elevated, but the lack of response such as the nil responses by luteal-phase females made statistics difficult; thus statistical restraint was applied. Flehmens and longer pre-flehmen responses (i.e. place and suck responses) were elicited in more than half of the follicular-phase females, whereas such responses were not seen with females tested in their luteal phase (Table 4).
Responses tabulated from repetitive testing of five different females (B,
J, P, S and T) during their follicular and luteal phases were statistically
compared. First, however, we tested both the data collected during the
follicular phase and that collected during the luteal phase for intra-phase
differences by one-way repeated measures of ANOVA and then pair-wise multiple
comparisons using the Student–Newman–Keuls test. For MO responses,
no intra-phase differences were observed for either follicular or luteal
phases [
2 = 36.5 with nine degrees of freedom (d.f.),
probability (P) < 0.001; difference of ranks (DR) =
13.5–39.5, q = 4.42–8.56]. For pre-flehmen responses, no
differences were observed during the follicular phase; however, several
significant intra-luteal differences were noted (2 = 41.0 with
d.f. = 9, P < 0.001; DR = 12.0–35.0, q =
4.50–9.84). Flehmen data revealed many non-responders; these zeros
precluded vigorous testing, but no significant differences were revealed
(2 = 20.0 with d.f. = 9, P = 0.018).
Subsequently, the responses of individual females during their luteal phase were compared with those during the follicular phases (Table 7). As analyzed by repeated measures of ANOVA on ranks followed by pair-wise comparison using the Student–Newman–Keuls procedure for all five females with five repetitive tests in each phase, all female elephants in the follicular phase exhibited a significantly higher frequency of chemosensory responses than females in the luteal phase for the MO and pre-flehmen responses (Table 7). No flehmens were observed to frontalin during the luteal phase (Table 6); however, one or two flehmens per session were often observed during the follicular phase (Table 5).
The four pregnant females tested for the first time with frontalin demonstrated statistically significant fewer MO responses (Table 8) than did the follicular-phase females (Table 9). The first tested pregnant females showed no flehmen responses (Table 8). Pre-flehmen responses for first-tested pregnant females were not different from responses from the single-tested follicular-phase females, but were significantly greater than those from luteal-phase females (Table 9). Likewise, the three triple-tested pregnant females (Table 10) demonstrated significantly higher levels of pre-flehmen responses than females in the luteal phase (d.f. = 33, P < 0.001, F = 12.5–29.9). Statistically, the triple-tested pregnant females demonstrated lower flehmen responses than follicular phase females (d.f. = 33, P = 0.05, F = 4.25).
Some noteworthy behavioral sequences following chemosensory responses were seen among follicular- and luteal-phase and pregnant females (Table 11). The sequential behaviors varied somewhat, depending on the estrous state or pregnancy. On two occasions, luteal-phase females demonstrated avoidance of frontalin by circling the sample. Pregnant females were considerably more apprehensive; during one of the two rumbling vocalization sessions, a female urinated and then performed a palatal pit contact response to a conspecific. Characteristically, during all but one assay of the pregnant elephants, palatal pit responses (either to self or to a conspecific) were observed. During one multiple palatal pit sequence, the initiator elephant checked the temporal gland of a conspecific (Table 11). The follicular-phase females were chemosensorily responsive, spent extended periods at frontalin samples, and performed penile and temporal gland checks on male elephants.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This paper presents evidence from bioassays of synthetic frontalin with conspecific Asian elephants demonstrating that frontalin is a putative pheromone in this species. Frontalin is released from a specific gland (the temporal gland) of male elephants during a specific physiological state (musth) and carries an intraspecies message to conspecifics, eliciting immediate chemosensory and behavioral responses. A differential spectrum of chemosensory responses and immediate, subsequent behavioral reactions by male and female elephants support the reception of a finely tuned chemical message. Tests with subadult and adult male and female African elephants evoke only sniffs and exhalations and no overt behaviors (T.E. Goodwin, B.A. Schulte and L.E.L. Rasmussen, unpublished data).
Pheromones are further defined as contributing `to evolutionary fitness or
mutual benefit of both sender and receiver'
(Meredith, 2000). Frontalin
fulfills this definition in the Asian elephant. The frontalin chemosensory
message from the musth temporal gland of older, more experienced, often
dominant male elephants gives a clear indicator to male (and female)
conspecifics of the sender's sexual state and social maturity. Other males of
equal status can choose to ignore such signals or on rare occasions may engage
in combat. Younger, smaller males, not releasing frontalin themselves, can
avoid costly fights with older, larger, more experienced males
(Rasmussen et al.,
2002). Females can assess maturity, physiological condition, and
reproductive fitness as older males stay in musth longer and secrete more
frontalin. Elevated chemosensory responses by follicular-phase females and the
lack of response by luteal-phase females reflect differential assessments that
mutually benefit male senders and female receivers. Apprehensive reactions and
elevated chemosensory responses by pregnant females indicate their recognition
of the advantage of not encountering aggressive musth males.
Frontalin as a pheromone also imparts important short-term social and reproductive messages that elicit immediate and evidential reactions and behaviors from both sexes. Older males exhibit awareness of frontalin by sniffing or checking, but only indifference follows. In contrast, younger subadult males proffer the same frequency of sniffs to frontalin but significantly more pre-flehmen and flehmen responses, and often vocalize or exhibit a variety of retreat postures (Table 3). Females also exhibit varied immediate responses that segregate according to their individual hormonal condition and reproductive state. Luteal-phase females, while not disinterested in frontalin, have a much lower response than follicular-phase females in all categories of chemosensory responses. While the olfactory responses of luteal-phase and pregnant females were similar in frequency, pregnant females were very responsive in the preflehmen category, exhibiting many check, place and suck responses, enabling identification of frontalin and probably its concentration.
These olfactory/vomeronasal organ responses were reinforced by sequential behaviors that suggested differential functionalities for frontalin. Subsequent or simultaneous behavioral responses, occurring after observed chemosensory responses to frontalin, were consistent within groups of similar hormonal phases (females) or maturity status (males). For example, luteal-phase females, after sniffing frontalin, exhibited immediate avoidance behavior (i.e. circling the frontalin sample) (Table 11). Pregnant females, with a higher frequency of sniffing, frequently rumbled after touching frontalin, whereas the checking of a conspecific's temporal gland after responding to frontalin suggests an attempt to identify the source of the chemical signal. In contrast, follicular-phase females performed numerous pre-flehmen and flehmen responses, at times followed by checking of the male penis or temporal gland, with no evidence of aversion.
The sexual and hormonal status differences in response to frontalin are
consistent with the different lifestyles of males and females. The male
lifestyle involves a more independent existence, greater wandering, and more
curiosity and boldness in sampling new odors and tastes
(Sukumar, 1989). In this
study, young males demonstrated the highest responsivity, especially related
to vomeronasal olfaction, and were more overtly dramatic in their avoidance or
repulsion to frontalin than either highest responding females (those in the
follicular phase or pregnant), or older experienced males who were indifferent
to moda musth secretions (Rasmussen et
al., 2002) and somewhat indifferent to frontalin. In the
tightly knit female society, females track each other's state of estrus
(Slade, 1999;
Slade et al., 2003).
Their responses to urine and urogenital secretions are influenced by their
hormonal status (Rasmussen and Schulte,
1998; Schulte and Rasmussen,
1999). After encountering frontalin (or older male TGS),
follicular-phase females respond with chemosensory responses and subsequently
with reciprocal palatal pit responses and vocalizations. Such behaviors
suggest inter-female transfer of information.
The two procedures for bioassays provide robust comparative data sets for defining frontalin as a pheromone of Asian elephants. For both sexes, bioassay data demonstrated that first tests (Tables 1, 4, 8 and 9) and repetitive testing (Tables 2, 5 and 10) gave similar results. Single tests are often preferred in behavioral studies to avoid pseudoreplication problems. However, to establish that a particular compound is a robust pheromone, it is necessary to demonstrate that elicited responses are not novel substance reactions. Repetitive testing of the same individuals confirmed the robustness of frontalin as a putative pheromone.
In examining pheromonal actions in elephants, their high cognitive
abilities and excellent long-term memories, especially regarding odors
(Rasmussen, 1995,
1999b), need to be considered.
For a meaningful study of captive male elephants, a personal life experience
history, including past and present musth status, hormonal levels, and past
associations that account for individual differences, is necessary to discern
possible functions. For example, one male OA1, although of similar age to the
other older adult males, is subdominant and is less socially mature as the
result of his particular life experiences; consequently, his responses for the
first two assays fit the pattern of a younger male. As the dominance dynamics
of the males changed, his responses fit into the adult grouping.
Comparison of the targets for musth male-released frontalin with that of
the urinary-derived female sex pheromone (Z)-7-dodecenyl acetate
demonstrates a clear difference in specificity of response (Rasmussen et
al., 1996,
1997;
Rasmussen, 1999a).
(Z)-7-Dodecenyl acetate elicits responses specifically from male
elephants, whereas frontalin elicits varying behaviors and chemosensory
responses from both males and females. Such a profile of directed behavioral
responses to the production of sex-specific compounds has been observed in
other mammals. For example, male mice release
3,4-dehydro-exo-brevicomin (an oxygenated terpene related to
frontalin) in their urine that elicits male aggression and female attraction
(Novotny et al.,
1985; Jemiolo et al.,
1991). This effect is further enhanced by co-secretion of
farnesene (Novotny et al.,
1990). In the future, we will examine whether any of the
substituted cyclohexanones (Perrin and
Rasmussen, 1994) in TGS potentiate frontalin-elicited behaviors
and determine whether several identified pheromonal ligand carriers (Rasmussen
et al., 1998,
2001;
Lazar, 2001; Lazar et
al., 2001,
2002) have roles in
effectively modulating these additional compounds in the ecological milieu of
the Asian elephant.
It is interesting to speculate on the remarkable coincidence that
frontalin, Z-7-dodecenyl acetate, and dehydro-exobrevicomin—all
previously identified as pheromone constituents in insects—also function
in strikingly similar roles in higher organisms. Most likely it is a
consequence of the comparatively limited biosynthetic capability of animals to
produce volatile compounds, with lipid and terpene synthons
(Francke et al., 1995)
being obvious choices for small molecular weight pheromonal ligands. Of
interest therefore is the source of frontalin in elephants. Is it synthesized
by males de novo or, more likely, is it transformed from
plant-derived terpenic precursors by microbial action in the fermentative hind
gut of the elephant and transported in the blood for release in the TGS?
Evidence for the latter scenario comes from the finding of frontalin in the
blood of musth males (L.E.L. Rasmussen, unpublished results). It will also be
interesting to determine the chiral form of elephant frontalin. Both (+) and
(-) forms may be active since the racemate supplied in the bioassays in the
current study is obviously functioning in responsive individuals.
In conclusion, we have demonstrated that the bicyclic ketal frontalin functions as a male-generated pheromone in the Asian elephant, eliciting a range of behaviors in conspecifics depending on their sex, age and physiological state.
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Acknowledgments |
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We thank Gary Jacobson and Jim Williams at the RCEC, Scott and Heidi Riddle at the Riddle's Elephant Sanctuary, and Roger Henneous, formerly of the Oregon Zoo, for their assistance in the bioassays. Biospherics Research Corporation and a travel grant from RCEC made this study possible.
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Accepted April 30, 2003
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