Beaver (Castor canadensis) responses to major phenolic and neutral compounds in castoreum

Journal of Chemical Ecology, VoL 20, No. 12, t994

BEAVER (Castor canadensis) RESPONSES TO MAJOR PHENOLIC AND NEUTRAL COMPOUNDS IN

CASTOREUM

B R U C E A. S C H U L T E , I ' * D I E T L A N D M O L L E R - S C H W A R Z E , I

R O N G T A N G , 2 and F R A N C I S X. W E B S T E R 2

I Department of Biology

'Department of Chemistry State Universi~ of New York

6¥)llege of Environmental Science and Forestry Syracuse, New York 13210

(Received March 21, 1994:, accepted July 18, 1994)

Abstract--North American beaver (Castor canadensis) mark their territories with castoreum, a chemically complex secretion from their castor sacs. The phenolic and neutral fractions of castoreum have been shown to elicit specific behavioral responses from beavers in a field setting. Our objective was to identify compounds/mixtures that evoked responses similar to those stimulated by castoreum. We assayed recently identified phenolic compounds, some phenolics that had been determined to be biologically active in previous studies, the neutral compound bomeol, and combinations of phenolic compounds, neutral compounds, and the two combined. Biological activity was measured by the elicitation and extent of specific responses and their strength (duration, frequency, and proportion of beavers responding). Generally, single compounds stimulated fewer responses than mixtures, A 26-compound mixture of phenolic and neutral compounds elicited responses in a similar proportion of trials as castoreum. However, responses to castoreum were stronger than to any synthetic sample. Further investigation of different measures of response, namely, elicitation, completeness, and strength, are deemed necessary to fully decipher the design of social odors.

Key Words--Castor canadensis, beaver, castoreum, communication, social odors, phenolic compounds, neutral compounds, territorial behavior, response measures.

*To whom correspondence should be addressed at Department of Chemistry, Biochemistry, and Molecular Biology, P.O. Box 91000, Oregon Graduate Institute of Science and Technology; Portland, Oregon 97291-1000.

3063

0098-0331194/i200-3063507.0010 0 1994 Plcnum Publishing Corporation

3064 SCHULTE ET AL.

I N T R O D U C T I O N

Species-specific mammalian olfactory signals, termed social odors or pheromones, contain diverse types of chemical compounds serving a variety of functions (Gorman, 1976; Jemiolo et al., 1985; Singer et al., 1976, 1986; Smith et al., 1985; Klemm et al., 1987: Belcher et al., 1988). Communicative scents often include numerous compounds that appear to play no role in the olfactory message. The remaining compounds may work together through addition, syn- ergism, redundancy, or suppression to construct the signal's meaning, which is elucidated typically by observing the responses of the receiving animals. Deci- phering the role or necessity of individual components in a biologically active fraction of a social odor requires extensive assays of compounds, singly and in mixtures.

The North American beaver, Castor canadensis, is an excellent model for field studies of mammalian social odors because beavers rely heavily on scent, but less so on visual or auditory communication; perform a highly detectable and stereotypical scent marking behavior; can be live-trapped and tagged for identification; are easily located and observed at close range; and are found in large enough populations for meaningful data acquisition. Beavers live in family units and are considered to be monogamous (Svendsen, 1989 and references therein). Their year-round territories are demarcated by mud mounds on the banks of their ponds. Beaver mounds are marked with urine and castoreum from the castor sacs, and possibly with anal gland secretion. While marking may serve multiple purposes, recent studies have supported a territorial function as primary (Houlihan, 1989; Welsh and Miiller-Schwarze, 1989).

Yet, we still have an incomplete understanding of the functional composition of beavers' territorial signals. For instance, are these signals individual identifiers, species-specific social odors, or pheromones that couple explicit mes- sages (e.g., warning, ownership) with specific responses (e.g., avoidance, scent marking)? Individual markers may be key compounds or more likely arrays of compounds in different ratios. Responses to synthetic odors that may represent an individual are difficult to predict without an understanding of the individual being mimicked. If a social odor, composed of redundant components, signals "beaver , " then many compounds or mixtures may evoke similar activity. The social odor hypothesis predicts mixtures should generally be more active than single compounds. However, if a territorial pheromone, consisting of one or a few specific compounds, exists, then most single compounds or mixtures from biologically active fractions should evoke little activity. The relative activity of compounds versus mixtures cannot be explicitly predicted without an understanding of the pheromone's chemical composition. Furthermore, pheromones and species-level social odors should elicit similar responses from year to year,

BEAVER RESPONSES 3065

whereas individual cues (and subsequent responses) may change on much shorter time scales.

Although beaver castoreum is a chemically complex secretion consisting of well over 100 compounds (Lederer 1946, 1950; Maurer and Ohloff, 1976), only specific chemical fractions evoke responses by beavers. Placement of whole or partial fractions of these secretions on human-made mud piles releases typical scent marking behavior (Miiller-Schwarze et al., 1983, 1986), which includes oriented sniffing from the water, visitation to the experimental scent mound (ESM), and overmarking of the ESM. By definition, land visits to the ESM are indicative of greater biological activity than oriented sniffing from the water. Beavers were attracted to ESMs containing the phenolic and neutral components of castoreum but not the carboxylic acids or amines (Svendsen and Huntsman, 1988). Several single castoreum compounds, especially phenolics, were active alone and in mixtures (Miiller-Schwarze and Houlihan, 1991; Miiller-Schwarze, 1992).

A primary goal of this research was the continued search for castoreum components that evoked similar responses (activity) as whole castoreum. Activ- ity was measured in the ability to elicit specific responses, the completeness of land visit responses to the ESM, and the strength of these responses. Therefore, recently identified and/or abundant phenolic compounds, the neutral compound bomeol, and progressively larger mixtures of phenolic compounds, a mixture of neutral compounds, and combined mixtures of the two were assayed. The compound 4-ethylphenol exhibited strong activity in a previous study (Mfiller- Schwarze and Houlihan, 1991) and was retested at four concentrations to further investigate its candidacy as a territorial pheromone. Our focus was on assaying phenolic and neutral compounds with the intention of better understanding the overall design of the castoreum odor signal in a territorial context.

METHODS AND MATERIALS

Study Area. All field experiments were conducted during May-September, 1989-1992, in Allegany State Park, Cattaraugus County, New York State, on an unharvested beaver population that had been live-trapped, ear-tagged, and observed since 1985. Sixteen to 20 beaver sites, consisting of one to several small ponds located on streams, were used for behavioral observations each year. The average family size was 5.5 animals (SD = 2.9, range 1-14).

Assumptions of Bioassay Design. Measuring the responses of the territorial resident to the odors or components of those odors from potential conspecific intruders is the basis of this bioassay design. The observed behaviors may reveal functionally relevant information, and any samples eliciting biological activity may truly play an important role in the social odor. Before attributing responses

3066 SCHULTE ET AL.

by beavers to the experimental stimuli, we examined our four basic assumptions about the continuity of behavior: (1) the responses of beavers did not vary significantly over time within a year, or (2) across years; (3) beaver responses to castoreum components were not the result of associative learning to the castoreum control; and (4) beavers from different family units (sites) responded similarly to the same samples. Previous studies have shown assumptions 1 and 2 to be acceptable (Miiller-Schwarze and Houlihan, 1991; MiJller-Schwarze, 1992). In 1989, assumption 3 was examined by observing the responses to 4-ethylphenol at sites that had not yet been exposed to castoreum. This compound was selected because it was biologically active in a 1987 study at Allegany State Park (Miiller-Schwarze and Houlihan, 1991). In the 1987 study, castoreum was assayed at sites prior to other samples. Comparing responses to 4-ethylphenol between the two years would allow us to test whether responses were the result of associative learning to castoreum. In regards to the final assumption, Houlihan (1989) demonstrated that neither family size nor the number of beaver sites along a stream were important variables in the responses of beavers to ESMs. However, other factors may contribute to response differences among sites. To test the null hypothesis that beavers at different sites responded to the same samples in a similar manner, beaver sites that had been used in assays of the same samples over the four years of this study were assembled into seven groups of three to nine sites each. Comparisons of the observed ("sniff from water" and land visit) and overnight land visit responses were made among sites within these groups.

General Methods. Beaver sites were surveyed for activity each spring prior to bioassays. Beavers were trapped with Hancock Live-Traps, weighed, and immobilized with a 1 : 2 mixture by volume of Rompun (xylazine) and Ketaset (ketamine) (0.67 and 6.7 mg/kg body weight, respectively). Sedated beavers were sexed (Young, 1936; Osbom, 1955; Schulte et al., in review), assigned to an age class (adult, 2-year-old, l-year-old, kit) based on weight and size, and individually tagged with anodized aluminum ear tags in unique color combinations for both ears. From 1985 to 1992, 245 beavers were tagged. Of the beavers observed during the experiments, 70% were recognizable by tags, including 86% of the adults. Untagged beavers were identified by distinguishing physical marks and assigned to an age class based on their relative size.

Stimulus Preparation. We assayed individual compounds and selected mixtures of compounds that have been identified as components of castoreum (Tang et al., 1993, in preparation). A few commercially available castoreum constituents used in a previous study (Mfiller-Schwarze and Houlihan, 1991)--4-ethylphenol, 4-propylphenol, salicylaldehyde--and the neutral compound bomeol were retested. The amount of each sample assayed and the composition of mixtures of phenolic and/or neutral compounds are given in Table 1. The ratio of compounds in mixtures was based on their ratio in a sample of 16 pooled,

BEAVER RESPONSES 3 0 6 7

TABLE 1. NAMES, ABBREVIATIONS (ABBR.), YEAR ASSAYED, AND SUMMARIZED

BIOACTIVITY OF SAMPLES USED IN FIELD BIOASSAYS, 1 9 8 9 - 1 9 9 2

Activily '~

Name Abbr. Year Sir Eli Corn

A. Single Compounds (rag/trial) Acetovanillone AC - - Bomeol (0.56; 0 .48/ BO 90; 92 I; I I; ! M; P 2-catechot (0.014) CT 89 I I 1 2,6-dimelhoxy~--methylphenol DC 90; 92 I; I I; I I; I

(0.11) 3,5-dimethoxyphenol DP - - 4-ethylguaiacol (0.83) EG 90 I I I 4-ethylphenol (0.691 EP 89 P I P

4-ethylphenol (0.032) E1 90 I I t

4-ethylphenol (0.32) E2 90 I I 1 4-ethylphenol (3.21 E3 90 1 I I 4-ethylphenol (321 E4 90 I I 1

4-(4 ' -hydroxyphenyl)-2-2-butanone HB - -

Indanol 1N - - m-cresol I0.023) MC 89 P I 1 4-methylguaiacol (0.033) MG 90 M P 1 o-cresol (0. I 1 ) OC 89 P I 1

4-propylguaiacol (0,025) PG 90 P 1 l

Phenol PH - - 4-propylphenol (0.37) PP 89 I 1 I Salicylaldehyde (0.025) SA 89 1 1 1

B. Mixtures BO + EG + MG + PG BG 90 1 I 1 O C + MC + CT + SA (4 phenols) FP 89 P P I FP + EP + PP (6 phenols) SP 89 1 I P

SP + PH + HB + IN + A C ( 1 0 TP 90 I I I

phenols) TP + G (13 phenols) TG 92 I 1 P TP + BG (13 phenols + bomeol) TBG 90; 92 I; P I; P I; P TP + EG + MG + PG + D C ( 1 4 P 9I ; 92 I; 1 I; I A ; P

phenols) P + B (14 phenols + borneol) PB 92 I I P

BO + 11 other neutrals/' N 91 M I P P + N ( I : I ratio) P + N 91 A A P P at 5-fold concentration 5P 91 M P P N at 5-fold concentration 5N 91 A M P 5P + 5N ( | : 1 ratio) 5P + 5N 91 A M A

3068 SCHULTE ET AL.

TABLE 1. CONTINUED

Activity"

Name Abbr. Year Str Eli Corn

C. Odor controls Castoreum (maximum response) CA 89-92 A A A Ethanol (minimum response) OH 89-92 I I I

D. Blank (no applied odor) BL 91; 92 I: I 1; 1 I; 1

"Activity -- not tested: Sir = strength of observed land visit is a composite bioactivity based on average duration of first land visit, number of land visits, and proportion of beaver visiting sample versus controls; Eli = elicitation of observed land visit compared proportion of trials in which land visits were observed to sample versus controls; Corn = completeness of overnight land visit is a categorial comparison of extent (none. paw, scratch, flat/remark) of modification to ESMs marked with a sample versus controls. See Table 2 for explanation of activity codes where I = inactive; P = potentially active: M = mildly active: A = active. Assayed amounts of mixtures were 1/30 gland equivalent, except for three 5-fold concentrates, with the ratio of compounds based on those in 16 pooled, male castor sacs (Tang et al., 19931.

1'The 12 neutral compounds were bomeol, benzaldehyde, benzyl alcohol, 6-methyl-l-heptanol. trans- and cis-linalool oxide, 4,6-dimethylheptane-l-ol, trans-pinocarveol, isopinocamphone, ver- benone, ( I R)-myrtenol. and 3,4-dimethoxy-acetophenone.

male beaver castor sacs (Tang et al., 1993). Four concentrations of 4-ethylphenol

(1000-fold range) and two concentrat ions (five fold range) of three mixtures

were also assayed.

Each year, ethanol and castoreum served as minimum and maximum

response elicitors, respectively. Ethanol was the solvent (usually 0.25 ml/trial)

for all samples, except the castoreum control in 1990-1992, which was pur-

chased as quill (dried beaver glands ground into small granules) from a trapping

supplier and presented in dry form. Responses to castoreum dissolved in ethanol

(1989) and to quill did not differ significantly. Blank (mud only) mounds were

used in 199t and 1992. Earlier studies showed no significant responses to either

ethanol or blank ESMs (Mi.iller-Schwarze et al., 1986; Mfi l ler-Schwarze and

Houlihan, 1991).

Stimulus Presentation. For the bioassays, an ESM was constructed by

scooping mud from the pond with a small container, held by gloved hand. The

ESM was approximately 30 cm diameter × 10 cm high and located about 50

cm from the water ' s edge at a bank where beaver were able to exit from the

water. A size 18 cork (3.6 cm top diameter) was placed in the center o f the

mound, even with the surface. The sample was applied to the cork with a

disposable pipet. In order to detect subtle disturbances to the mound overnight,

one-half o f a tongue depressor was placed vertically into the mound (as by


Svendsen, 1980) adjacent to the cork. The placement of the tongue depressor does not interfere with beaver inspecting or remarking the mound.

Experimental Design. A minimum of eight trials and a maximum of 16 trials at different sites were performed for each sample during a single season. A trial was equivalent to a single-day, single-site assay. Samples were randomly assigned to beaver sites in 1989 (except 4-ethylphenot) and 1992 using a random number table. In 1990 and 199l, a Latin square design structure was imple- mented to balance the testing of the chemically similar samples over time at different sites. Beaver sites were grouped by proximity in the same watershed into clusters of four (1990) or three (1991) sites. Samples were also grouped by fours or threes based upon their similarity in chemical composition or type (i.e., yielding a 4 x 4 or 3 x 3 Latin square). The matrix size was determined by the number of available observers each season. Control trials were run at the beginning and end of assaying the samples in each square.

Behavior Observations. Behavioral bioassays were performed from June to September in 1989 and May to August in 1990-1992, following the procedures described by M/iller-Schwarze and Houlihan (1991). The ESM was built and the scent applied at about 1700 hr, generally 0.5-1 hr before the first beaver emerged. Each beaver was observed from the time it emerged until nightfall (2000-2130 hr, except in September 1989:1900-2000 hr). A computer program created by P. Houlihan and modified by C. Sack for the Tandy 102 portable computer automatically records the time of each behavior entered. A field note- book and a stopwatch were available to record observations in case of computer failure. When one or more beavers approached the sample, observations were focused on these individuals (i.e., focal location). An exhaustive ethogram was created, but the behaviors of greatest interest included: swim (fast, slow), float, tail-slap, dive, sniff from water (general or directed at ESM), land visit, and activity at the ESM (sniff, scratch, mark). Weather conditions, such as precip- itation, air temperature, and wind direction, and unusual observations were noted.

The ESM was checked the next morning for signs of visitation during the night, separated into four categories: (1) intact; (2) paw prints on an otherwise undisturbed ESM; (3) scratch marks, movement of the applicator, and/or removal of the cork; and (4) flattening or marking (fresh castoreum odor) of the mound. Activity at the ESM that could be attributed to other animals (e.g., whitetail deer, raccoon) was discarded. Marking was discernible by sniffing the mound and an area of 50 cm radius around the ESM.

Format of Data. For each year of the study, biological activity was determined by comparing responses to each sample with the responses to castoreum (maximum response control) and to ethanol (minimum response control). The responses to samples was also compared to the results from the controls pooled over the four years.

3070 SCHULTE ET AL.

The data were organized into two general types: observed responses and overnight land visits. The observed responses were sniffing from the water (directed towards and within 5 m of ESM) and land visits. In a sniff response, beavers orient toward the ESM and noticeably sniff by raising their lower jaw slightly out of the water. Land visits occurred when a beaver exited the water, moved directly to the ESM, and sniffed and/or contacted the ESM. Three numer- ically continuous parameters, and one categorical parameter, of the observed responses were used: (1) average duration in seconds per beaver responding per trial; (2) average number of responses, calculated as the number of sniffs or land visits per beaver responding per trial; (3) the proportion of the number of beaver responding to the total number of beaver seen per trial; and (4) classifying the trials as no response, sniff from water only, or land visit. Because beavers

live in family units, duration of land visits was determined both for the first individual to respond and for all responding members of the family unit combined. The three continuous parameters were calculated by first using all trials, then a second time by including only those trials in which responses were observed. During observations of land visits, beavers would often sniff the ESM without flattening or overmarking it. Therefore, an observed land visit does not necessarily include a scent-marking response. Responses by kits were not included in the observed analysis.

The data from overnight land visits were analyzed by the categories of response (intact, paw, scratch, flat/re-mark). Each trial was placed in one category only, representing the most complete response recorded. If a beaver left no trace of its presence on or near the ESM, then the ESM was labeled intact, even if a beaver had visited the ESM during the observations. This maintained the independence of the observed and overnight measures of land visit response. Trials in which no beaver were seen were included only in the overnight data set, and only if beaver were determined to still reside at the site.

Statistics. For each response measure, castoreum controls were compared across all four years using analysis of variance (ANOVA) for the continuous data and the log likelihood goodness of fit test (G) for the categorical data. Because the assumptions of normality and equality of variance for ANOVA were often not met with the raw data sets, Kruskal-Wallis tests were performed by year. Each sample was compared by Wilcoxon's rank sum test to its maximum and minimum controls from the same year. Contingency table analysis was used for comparisons of the categorical data. The Yates correction in 2 x 2 contingency tables and the Williams correction for larger tables were utilized to provide a conservative test of the null hypothesis (Sokal and Rohlf, 1981). Exact P values (Mehta and Patel, 1992), calculated for these likelihood ratio tests, agreed quite well with the P values approximated from the G statistic (i.e., the conclusions drawn from the data analyses were the same).


The Statistical Analysis System (SAS, Cary, North Carolina) and the BIOM package of statistical programs (Rohlf, 1987) were used for all data analyses.

Bioactivi~ Criteria. As minimal requirement for activity, we stipulated that the responses to a sample were statistically different (c~ = 0.05) from the responses to the ethanol control. Bioactivity was determined by considering the proportion of trials in which a particular response was observed (elicitation) or for overnight land visits the extent to which the ESM was disturbed (completeness) and the duration, frequency, and proportion of beaver sniffing from the water or visiting the ESM (strength) for the observed responses. Each sample was categorized into one of four activity levels for each response measure (Table 2). Bioactivity measures of completeness and strength included a component of response elicitation because trials with no responses were considered as values of zero to ensure appropriate sample sizes for the statistical analyses (Table 1).

RESULTS

Examination of Assumptions of Bioassay Design. Responses by beaver to the experimental samples did not appear to change across time, either within a season or between years. All regressions of sniff from water and land visit responses to the castoreum control against time were nonsignificant (all P > 0. t3). The responses to castoreum across the four years also did not differ statistically, although responsiveness appeared to diminish in 1992 (Table 3A). In addition, the preparation of the castoreum control (ethanol solvent in 1989 versus dry quill in 1990-1992) had no effect on the response. Responses to the ethanol control did not differ within or between years (Table 3B). Responses to ethanol also did not differ from those to the blank ESMs. Since the beaver did not respond differently to the controls in the four years, their disposition to respond to all other stimuli was assumed not to differ either.

Animals may respond to a placed sample because of its association in time or space with a biologically meaningful chemical signal. We tested this by

TABLE 2. ACTIVITY LEVELS ASSIGNED TO SAMPLES (TABLE 1) BASED ON PAIRWISE

WILCOXON RANK SUM TESTS (COMPARISON ERROR RATE = 0.05) WHERE IDENTICAL

LETTERS INDICATE NO STATISTICALLY SIGNIFICANT DIFFERENCE

Ethanol Sample Castoreum Activity level

a a b Inactive (I) a ab b Potentially active (PA) a b c Mildly active (MA) a b b Active (A)

3072 SCHULTE ET AL.

TABLE 3. RESPONSE BY TRIAL TO CASTOREUM AND ETHANOL CONTROLS a

Responses by trial

Observed ~' Overnight ~'

None Sniff LV ' Intact Paw Scratch Flat

A. Castoreum 1989 3 1 4 1 1 2 4

1990 3 0 8 I 0 I 11

1991 2 3 10 3 4 3 5

1992 3 2 6 4 4 0 3

1989-1992 11 6 28 9 9 6 23

B. Ethanol 1989 7 1 0 7 0 0 I

1990 11 0 0 11 0 0 0

1991 11 1 0 10 3 0 1 1992 6 2 1 10 0 0 0

1989-1992 35 4 1 38 3 0 2

"Values in boxes are number of trials for each response category. For castoreum (CA) the years were not statistically different using the G (Williams) value for contingency table analysis and exact or nearly exact (Monte Carlo) P values for observed (G = 4.96, df = 6, P = 0.60) or for the overnight visitation (G = 16.8, df = 9, P = 0.06); the same was true lot ethanol, observed (G = 3.07, df= 6, P = 0.37), Overnight (G = 4.13, df= 6, P = 0.10); fi~r ethanol the paw + scratch categories were combined because of zeros. For each year, CA was significantly different from OH, as were the combined totals from the tour years (combined: observed G = 43.2, df = 2, P < 0.1301; overnight G = 48.8, df = 3, P < 0,(X)I),

~'See text for complete explanation of categories. ' LV = land visit to ESM. Land visits may have been preceded by sniff's, but trials in the sniff column had no land visits.

assaying the single compound 4-ethylphenol (EP) both before any castoreum samples were assayed (1989, this study) and after assays with castoreum conducted at Allegany State Park in 1987 by Mtiller-Schwarze and Houlihan (1991). The similar levels of response to EP between the two studies (if anything slightly stronger in 1989) suggested that association with the castoreum control was not the cause of the responses. In the 1987 study, beavers were observed to visit the ESM in 10% of the trials compared to the 12.5% recorded in 1989. Over- night visitations were made in 25% of the trials in 1987 compared to 62.5% in this study. Thus, beavers did not respond to 4-ethylphenol as a result of association with castoreum in the sequence order of assays. If these results were generalizable for all samples, then the order of the sample relative to castoreum in the experimental design should not have affected the response of the beaver.

Furthermore, beavers from different sites responded similarly to the chemical samples. While beavers from separate sites did vary in their amount and

BEAVEn nESPONSES 3073

degree of responsiveness, responses were not statistically different among sites for most site groups. For instance, of the 16 sites used most frequently through- out this study, beavers at one site exhibited a reduced level of response, while beavers at a second site displayed greater responsiveness. Beavers at the remaining 14 sites behaved similarly to the same samples both within and among years. Therefore, the bioactivity of samples cannot be attributed to the response char- acteristics of the beavers at the sites where the assays were performed, but rather should be generalizable across beavers from most sites, at least in our study area.

Obsen,ed Sniff from Water Responses. Responses to the samples differed mainly in their probability of evoking sniffing and not the strength of the response (i.e., duration or frequency of sniffs or the proportion of beavers sniffing). Beavers sniffed ethanol in only four of 40 trials (10%) compared to 31 of 45 (69%) for castoreum (G = 30.5, 1 df, P < 0.001). Considering only trials in which sniff responses were observed, the average duration of sniffing ESMs containing ethanol was nearly as long (9.1 sec + 5.4) as castoreum (11.2 sec + 1.1). The number of responses to ethanol were two few to statistically com- pare with responses to castoreum (Figure t). Sniffing was elicited in at least 50% of the trials by only six samples--one single compound (EP) and five mixtures: EP 50%; six-phenol mixture (SP) 50%, 12-neutral mixture (N) 56%, 12-neutral mixture at fivefold higher concentration (5N) 78%, 26-compound mixture of phenolic and neutrals (P + N) 56%, and 26-compound mixture at fivefold higher concentration (5P + 5N) 67%. The mean sniff duration ranged

(it) Comlmuads (b) Mixtures

4 ~I[3 ~ 3 3-3 4 3 3 5 31

~lll l l l l l l l l l l l l ~ lo 4 4

io

,d

o 4 6 } 0 1 2 1 3 1 4 ]4 1526

S A M P L E S S A M P L E S

FIG. 1. Duration of sniffing from water by beaver to compounds, mixtures, and controls for trials with responses only. Compounds are ordered based on activity. Mixtures are ordered by increasing number of compounds in sample (shown along abscissa in b). Bars are one standard error. Sample sizes shown above error bars. Samples lacking error bars had only one value: samples with (-) had no response. See Table 1 for key to abbreviations of samples.

3074 SCXUL'rE ET AL.

from 4 to 17 sec with greater means having correspondingly high variations (Figure 1). While very few samples evoked sniffing in a majority of the trials, once initiated, the duration of sniff responses by beavers were not highly dis- criminatory among the samples. The same was true for frequency of sniffs and the proportion of beaver sniffing (results not shown).

Observed Land Visits. During land visits beavers directly sniffed the ESM and surrounding area, often followed by beavers standing upright and rotating their heads while sniffing the air. On some instances beavers scratched at the ESM, occasionally flattening and/or overmarking it.

Most of the samples triggered only few or no observed land visits. Hence, determination of bioactivity based upon strength of the observed land visits (Table 1) was made by including all no response trials (e.g., duration of land visit equal to zero) in the analyses. Five mixtures [four phenolics (FP), P + N, 5P, 5N, 5P + 5N] were visited in 30% or more of the trials. Only one single compound, 4-methylguaiacol, evoked a land visit in more than one trial (three trials, 23 %). Land visits to 4-ethylphenol (EP; E I-E4; concentrations and years pooled) were observed in three of 52 trials (6%).

Typically, land bouts lasted 20-45 sec, Only the mean duration of first land visits are shown (Figure 2) because land visits occurred just once per trial to each of the compounds and to most of the mixtures (Figure 3). Thus, there were no important differences in the duration of the first land visit versus all land visits (i.e., individual versus family response). On average, less than 50% of the beavers observed per trial responded to the sample (Figure 4). The two exceptions were to castoreum and a single trial with a land visit to 4-propylguaiaco! (PG).

(a) Compounds (b) Mixtures

IOO. IO0

80 ' 4 4 S O 3 7O ~ 28

g eo; i

'itllxlmm 50.

• 4 0 7

, 30 2 3

, , v , v 1 , , , , , , , , , v i

0 4 6 10 12 13 14 14 15 26


FIG, 2. Duration of first land visit by beaver to compounds, mixtures, and controls for trials with responses only. Samples ordered and format same as in Figure 1. Five compounds are grouped as SG5 = CT, E2, E4, PP, and SA. Sample sizes (above error bars) differ from Figure 1.


(I) Compousds (b) Mtx~t'es 2 2

0 4 6 10 12 t3 14 14 15 26


FIG. 3. Average number of laud visits (LV) by beaver to compounds, mixtures, and controls for trials with responses only. Samples ordered and format same as in Figure 1. Sample sizes and abbreviations as in Figure 2.

(u) Compounds 0b) Mixtures

o.$1 0.8 ~

o.6

' l i J l , i l l w i l r ~ ~ i i J 0.0 . . . .

o 4 6 1 0 1 2 1 3 1 4 14 1526


FIG. 4, Proportion of beavers making land visits (LV) to compounds, mixtures and controls for trials with responses only. Samples ordered as in Figure 1. Sample sizes and abbreviations as in Figure 2.

For the three mixtures tested at two concentrations (N, 5N; P, 5P; P + N, 5P + 5N), no significant differences were found in the observed responses by beavers between any of the concentration pairs. However, both the number of land visits and their durations in response to the high concentrations of the phenolic compounds (5P), neutral compounds (5N), and the two combined (5P + 5N) were more similar to castoreum than the responses to any other samples (Figures 2 and 3).

Relationship of Sniffing to Observed Land Visit. In general, the observed response variables were not linearly correlated (Table 4). However, the response

3 0 7 6 SCHULTE ET AL

TABLE 4, CORRELATION MATRIX OF OBSERVED RESPONSE MEASURES, 1 9 8 9 - 1 9 9 2 ~

Response Sniff Sniff Sniff First LV LV LV

variables duration number Pro duration number Pro

SF Dur 1.00 0.16 -0.09 0.16 -0.04 -0, 10 SF Num 0.22** 1,00 0.08 -0.16 0.50** 0,28 SF Pro -0,04 0.16 1.00 0.04 0.25 0.77** LV Dur 0,24 -0.04 0.09 1.00 0.02 0.29* LV Num -0.03 0.49** 0.41 0.15 1,00 0.22 LV Pro -0.04 0.31" 0.81"* 0.32* 0.32* 1.00

"Sniff (SF) = sniffing from water; LV = land visit; For each response type, duration (Dur) and number (Num) of responses, plus the proportion (Pro) of beaver responding/seen was determined, Only trials in which a sniff from water or land visit were observed are included in this analysis. Values in the top half of the matrix are Pearson product-moment correlations. Spearman rank correlation coefficients are shown in the bottom half of the matrix. The only high correlations are between the duration of first and all land visits, and between the proportion o f beavers sniffing and making land visits. Sample size: N = 137 for sniffing from water and N = 70 for land visit variables. For tests rejecting the null hypothesis that the correlation coefficient = O: *P < 0.05; **P < 0.001.

variables that were highly correlated were between sniff from water and land visit variables and not within either type. Specifically, the proportion of beaver from a family that made a land visit was positively correlated with the proportion of beaver sniffing (r = 0.77, P = 0.0001). Sniffing preceded land visits in 71% of the trials. Thus, most beavers that made a land visit sniffed from the water first in an observable fashion. In addition, the average number of land visits was correlated with the average number of sniffs from the water (r = 0,50, P

= 0.0003). The average number of sniffs and land visits per beaver was often just one in any trial, which may explain this correlation.

Overnight Land Visits, Strength (as defined by duration, frequency, and proportion of beaver visiting) of the overnight land visits could not be determined. Based on only the proportion of trials in which the condition of the ESM overnight was categorized as intact, pawed, scratched, or flattened/overmarked (completeness), the activity of the samples was determined (G statistic, 3 df): I0 compounds and three different mixtures were inactive; two compounds (EP; BO92) and nine mixtures were partially active; one compound (BO90) and no mixtures were mildly active; and no compounds and two mixtures (P, 5P + 5N) were active (Table 1). Three different mixtures were visited in a majority of their trials: P (88.9%) and 5P (55.6%); 5N (66.7%); P + N (66.7%) and 5P + 5N (55.6%).

Castoreum evoked overnight land visits in 38 of 47 trials (81%), while ethanol elicited land visits in only five of 43 trials (11.6%). In 29 trials, there were no overnight land visits to blank ESMs. Of the single compounds, only


4-ethylphenol (EP) was visited in over half its trials (5/8, 62.5%), However, responses to the four concentrations of EP tested the next year (1990) elicited fewer overnight land visits (18-37% of the trials). There was no pattern of response related to concentration.

Responses to borneol (BO90, BO92) showed the greatest contrast in the observed versus the overnight measures. Beavers were observed to visit borneol only once in 22 trials (4.5%) and only sniffed from the water in four trials. Yet, borneol was visited overnight in 10 of 27 ovemigfit trials (37%), flattened in four cases, and ov.ermarked three of those times.

In 1992, the responses by beavers to all of the samples, including castoreum, diminished, possibly because it was an exceptionally wet season. The responses to each sample were compared to those elicited by the controls over all four years of the study (castoreum, 1989-1992; ethanol, 1989-1992). Based on the degree of manipulation to the ESM overnight, the 14-phenolic mixture (P92) and borneol (BO92) were potentially active and other samples were inactive. Comparing the experimental samples from 1989 through 1991 to the pooled controls did not alter any of the results.

Bioactivi~ of 14-Phenol and 12-Neutral Mixture. The responses to the 26- compound mixture (pooling responses to the two concentrations) and to castoreum (1989-1992) were compared. The proportion of trials in which both observed and overnight responses were elicited by the 26-compound mixture was statistically indistinguishable from the same proportions in responses to castoreum (G statistic, 1 df, both P > 0.10), This was true for only this mixture. In addition, for trials with an overnight land visit, the extent of ESM modification (paw, scratch, flat) overnight in response to this mixture or castoreum did not differ significantly (P > 0.90, G = 0.09, 2 df). However, observed land visits to the 26-compound mixture were shorter (P = 0.02, Wilcoxon), fewer in number (P = 0.01), and made by a smaller proportion of beavers (P = 0.005) than land visits to castoreum.

DISCUSSION

A primary objective of this study was to identify castoreum compounds and/or mixtures of compounds that elicited scent-mark inspection behavior similar to that evoked by castoreum, A synthetic mixture of 26 castoreum constituents elicited land visit responses to the ESM in a similar proportion of trials as the castoreum control. However, observed land visits to this mixture (or any other sample) relative to castoreum were shorter in duration, fewer in number, and performed by a smaller proportion of beavers. Yet, the degree of modification of the ESM was not significantly different between ESMs marked with the 26-compound mixture and those with castoreum. The 26-compound mixture

3 0 7 8 SCHULTE ET AL.

may have provided a clearer or simpler signal that required less inspection to elicit particular responses than the more chemically elaborate castoreum. This may be analogous to the responses observed to castor fluid from familiar resident versus less familiar nonresident beaver (Schutte, 1993). In Schulte's 1993 study, beaver made shorter land visits to ESMs marked with the castoreum from the resident adult male than ESMs marked with castoreum from nonresident adult males. Thus, perhaps the 26-compound mixture is part of a recognizable, core beaver signal, yet lacking individualistic qualities that may evoke longer investigation.

The assumptions of behavioral continuity in time and space for our field studies conducted over several years at Allegany State Park appear reasonable. Our assays performed at numerous sites yielded results that were not affected by the order in which the samples were tested. Biological activity of the controls within a season and among years varied more in the range of behaviors elicited than in the relative amount of responses. At no time did beavers respond more to ethanol or blank ESMs than to castoreum. However, some intriguing differences in responses over time were apparent. For instance, the compound 4-ethylphenol (EP), shown to be active in a previous study (M/iller-Schwarze and Houlihan, 1991), evoked a strong sniff from water response and some scent- marking behavior by beavers in the 1989 season of this study. Yet, the four concentrations tested in 1990 were generally inactive. Active samples (i.e., potential pheromones) should stimulate responses across both time (e.g., within a season or between years) and sites. Failure to do so may indicate a novel compound response, or more interestingly, some temporal-spatial significance to the meaning of the signal. The likelihood that many of the compounds in castoreum are diet-related, and therefore transitory, may be very important in this regard.

We offer no support for a single compound serving as a territorial signal, yet many more compounds need to be assayed. In agreement with the social odor hypothesis, mixtures were more active than single compounds, and larger mixtures were more active than smaller ones, with some exceptions. Methods for evaluating overall bioactivity will be discussed in a separate paper (Schulte et al., in review).

Mixtures with more compounds may have stimulated activity in more trials either because the odor image (Albone, 1984) was enhanced, the compounds carried a redundant message, or the different compounds played distinct roles in the signal (or some combination). The odor image concept suggests a gestalt process of information transfer in that compounds do not play particular roles by themselves, but together they produce a meaningful signal (Beauchamp et al. , 1976). Redundant compounds may form a clearer signal and thereby increase detectability (Krebs and Dawkins, 1984) and subsequent responsiveness (i.e., elicitation) but not affect the completeness or strength of the response. Addi-


tionally, specific tasks may be carried out by certain compounds, or types of compounds, in the transmission of the message (Albone, 1984). For instance, some more volatile compounds may attract attention, while other components, possibly with longer retention times on the substrate, provide more detailed information (Alberts, 1992).

There was some hint of this latter pattern in the results. The phenolic, 4-ethylphenol, elicited more sniffing from the water than the neutral compound bomeol; however, overnight land visits to bomeol were more frequent and more complete than those to 4-ethylphenol. ESMs marked with borneol may have been chanced upon during foraging bouts, rather than directly attracting beavers from the water. If certain types of compounds (e.g., neutral versus phenolic compounds) play specific roles in signal transmission and/or meaning, then differences in the type of responses (i.e., sniff versus land visit) might be expected. Yet, responses to the mixtures of 12 neutral compounds (N, 5N) were not very different from those to the 14-phenolic compounds (P, 5P). Two phenolic compounds (EP, MG) and phenolic mixtures (SP, 5P) did evoke some long bouts of sniffing, but sniff responses to these samples were highly variable. Furthermore, some of the "inactive" phenolic compounds and mixtures, plus the mixture of three guaiacols and borneol (BG), stimulated sniffing but very few to no land visits (observed or overnight), More information on responses to neutral compounds is needed to examine their role in the castoreum signal and to determine if responses to neutral and phenolic compounds contrast in biologically relevant ways.

A complex secretion such as castoreum may serve several functions either simultaneously or over time. This olfactory fingerprint may identify individuality without coding for species, age, sex, status, or other information separately (e.g., Voznessenskaya et al., 1992). Understanding the signal structure and message in beaver scent mounds may help to reveal the specific function(s) of marking behavior. Separating the response components of elicitation, completeness, and strength may be helpful in deciphering the meaning and chemical composition of this signal. Elucidating the potential role of scent marking in beaver dispersal, territory maintenance, and mating behavior could be greatly beneficial in improving our ability to manage beaver populations and wetlands and in furthering our knowledge of the chemical ecology of mammalian social odors.

Acknowledgments--We thank the management and staff of Altegany State Park Ibr their cooperation in our study. Thanks go to P. Houtihan and C. Sack for the creation and modification of the behavioral recording and analysis programs. K. Bakinsky, T. Brockway, C. Calamita, S. Heerkens, M. Marion~ J. Orlowski, M. Rehberg, R. Robichaud, C. Rubeck, B. Speicher, L. Sun, and T. Schwender assisted with the field research. D. Schulte helped in innumerable ways. Gratitude is extended to Drs. L. E. L. Rasmussen, S. V. Stehman, S. A. Teale, and L. L. Wolf for their critical evaluations of the research design, statistical analyses, and manuscript. SUNY College of

3080 SCHULTE ET AL.

Environmental Science and Forestry furnished a field camp at Allegany State Park. Support |or this research was provided by National Science Foundation grant BNS-8819981 to D.M.S. and F.X.W.. and a grant to B.A.S. from the Theodore Roosevelt Fund.

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Beaver (Castor canadensis) responses to major phenolic and neutral compounds in castoreum