Smelling Wrong: Hormonal Contraception in Lemurs Alters Important Female Odour Cues

Smelling wrong: Hormonal contraception in lemurs alters important female odour cues

Jeremy Chase Crawford1, Marylène Boulet1, Christine M. Drea1,2*

1 Department of Evolutionary Anthropology, Duke University, Box 90383, Durham, North Carolina, 27708, United States of America

2 Department of Biology, Duke University, Durham, North Carolina, United States of America

* To whom correspondence should be addressed: .

S1. SUPPLEMENTARY METHODS & RESULTS

(a) Female access to males and semiochemistry

Because female access to males differed between the intact and contracepted conditions, we wanted to draw attention to two lines of evidence that our housing conditions did not influence the results.

The first line of evidence derives from observing continued olfactory communication and some level of social interaction between separated, but adjacently housed animals (figure S1).Although most intact females did not have physical access to adult males, they could see, smell, hear, and even touch their former male groupmates at all stages of the study. Adult members of the opposite sex that typically would be housed together (as was usually the case in our prior olfactory studies: Scordato et al. 2007: Charpentier et al. 2008, 2010; Boulet et al. 2009, 2010) were housed in adjacent indoor/outdoor enclosures, separated only by chain link fencing (figure S1).

The second line of evidence derives from various analyses showing that variation in social housing at the Duke Lemur Center has no effect on semiochemical profiles. A prior analysis in Charpentier et al. 2008 (appendix S2) showed the absence of housing effects on male chemical distances relative to other males. The housing conditions tested included the following: alone, male-only groups, one-male groups, and multimale-multifemale groups.A prior analysis in Boulet et al. 2009(supplementary table 1) likewise showed the absence of housing effects on female chemical distances relative to other females. The housing conditions tested for those intact females included the following: alone and multimale-multifemale groups. Here, we complement prior analyses more specifically by comparing chemical data obtained from 13 intact females, all of which had been sampled at the same time of year within the breeding season. At that time, eight had been housed with males and five had been housed without males. We tested the chemical data from these two groups of females, using Wilcoxan rank-sum tests, and found no effects of the two housing conditions on the chemical complexity of labial secretions. Specifically, odorant samples obtained from females in these two conditions did not differ in their Richness (Z = -1.24; p = 0.21), Shannon Index (Z = -1.54; p = 0.12) or Simpson Index (Z = -0.51; p = 0.61).Therefore, under the present housing conditions, semiochemistry was consistent in female lemurs, regardless of their access to males.

Figure S1. Two male ring-tailed lemurs, housed at the Duke Lemur Center in adjacent compartments separated by chain-link fencing, are actively engaged in a ‘stink fight.’ Stink fights (Jolly 1966) involve the transmission of olfactory information when males waft their odour-impregnated tails at one another. Photo provided courtesy of David Haring.

(b) Endocrine assays

Hormones were assayed using commercially available radioimmunoassaykits (Diagnostic Systems Laboratories, Webster, TX or Diagnostic Products Corp., Los Angeles, CA). All of the samples were processed together to avoid inter-assay variation. The OHP assay has a sensitivity of 0.10-40 ng/ml using a 100µl aliquot, with an intra-assay coefficient of variation (CV) of 8.44%. The A4 assay has a sensitivity of 0.13-10 ng/ml using a 50µl aliquot, with an intra-assay CV of 11.21%. The T assay has a sensitivity of 0.05-25 ng/ml using a 50µl aliquot, with an intra-assay CV of 6.3%. The E2 assay has a sensitivity of 5-1000 pg/ml using a 200µl aliquot, with an intra-assay CV of 7.95%. Assay cross-reactivities have been reported elsewhere (Drea 2007).

(c) Chemical assays

We analyzed odorant samples using gas chromatography-mass spectrometry (GCMS) following previously published procedures (Scordato et al. 2007; Charpentier et al. 2008). Briefly, we extracted thevolatile compounds with methyl tert-butyl ether, concentrated the extracts under compressed nitrogen flow (to 50-70µl), and added 5 µl of an internal standard (hexachlorobenzene, 1 mg/mL). We analyzed the samples with a Shimadzu GCMS-QP2010 instrument, equipped with a Shimadzu AOC-20 series autosampler(Shimadzu Scientific Instruments).

(d) Indicator Species Analysis

To determine if specific compounds could be used as predictors of a treatment group, we performed Indicator Species Analysis (ISA; PC-ORD; Dufrene & Legendre, 1997; McCune et al. 2002). ISA is traditionally used by ecologists to determine if species can be reliable indicators of certain environmental parameters. After analyzing relative abundances of species between two or more a priori groups, ISA provides an “indicator value” (IV), which estimates the degree to which each species acts as an indicator of a particular group. In the case of our two treatment groups, we considered individual semiochemicals as species. We also considered isomers as different semiochemical species (e.g., 1-hexadecanol a and b, Table S1). An IV of zero identifies species that are not representative of any treatment, whereas an IV of 100 identifies species that represent a specific treatment with no chance of error. ISA can also be used to evaluate each IV for statistical significance (p-value) using a randomization technique. Our results for this analysis are provided in tables S1 and S2.

(e) Analysis of odour-gene covariance

In previous studies (Charpentier et al. 2008; Boulet et al. 2009), classes of pairwise genetic distances between dyads were roughly equal in both sample size and range of genetic distance, but that was not the case here, given our smaller sample size. Consequently, we first performed the PERM analysis on categories that were roughly equal in range of genetic distance (as reported in the results section); we then repeated the analysis using categories that were roughly equal in sample size and replicated the previous results (data not shown).

REFERENCES

Boulet, M., Charpentier, M.J.E., & Drea, C.M. 2009 Decoding an olfactory mechanism of kin recognition and inbreeding avoidance in primates. BMC Evol. Biol.9, 281. (doi:10.1186/1471-2148-9-281).

Boulet, M., Crawford, J.C., Charpentier, M.J.E, & Drea, C.M. 2010Honest olfactory ornamentation in a female-dominant primate. J. Evol. Biol. (doi:10.1111/j.1420-9101.2010.02007.x)

Charpentier, M.J.E., Boulet, M., & Drea, C.M. 2008 Smelling right: the scent of male lemurs advertises genetic quality and relatedness. Mol. Ecol. 17, 3225-3233. (doi:10.1111/j.1365-294X.2008.03831.x)

Charpentier, M.J.E., Crawford, J.C., Boulet, M. & Drea, C.M. 2010Message ‘scent’: lemurs detect the genetic relatedness and quality of conspecifics via olfactory cues. Anim. Behav.doi:10.1016/j.anbehav.2010.04.005

Drea, C.M. 2007 Sex and seasonal differences in aggression and steroid secretion in Lemur catta: Are socially dominant females hormonally ‘masculinized’? Horm. Behav. 51, 555-567. (doi:10.1016/j.yhbeh.2007.02.006)

Dufrene, M. & Legendre, P. 1997 Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecol. Monogr.67, 345-366.

Jolly, A., 1966. Lemur behavior. Chicago: University of Chicago Press.

Scordato, E.S., Dubay, G., & Drea, C.M.D. 2007 Chemical composition of scent marks in the ringtailed lemur (Lemur catta): glandular differences, seasonal variation, and individual signatures. Chem. Senses32, 493-504. (doi:10.1093/chemse/bjm018)

McCune, B., Grace, J.B., & Urban, D.L. 2002 Analysis of Ecological Communities. Gleneden Beach, OR: MjM Software Design.

Table S1. Compounds present in the labial secretions of ring-tailed lemurs that predict the intact condition. Compounds are grouped by classification and ordered by molecular weight (m.w.). The classification and/or molecular weight forsome compounds were unknown (U). Asterisks denote compounds unique to the intact condition. Isomers are distinguished by the letters a-l.

Table S2. Compounds present in the labial secretions of ring-tailed lemurs that predict the contracepted condition. Compounds are grouped by classification and ordered by molecular weight (m.w.). The classification and/or molecular weight forsome compounds were unknown (U). Asterisks denote compounds unique to the contracepted condition. Isomers are distinguished by the letters a-e.