Journal of Chemical Ecology

, Volume 38, Issue 10, pp 1318–1339

Scent Chemicals of the Brushtail Possum, Trichosurus vulpecula

Authors

    • School of PharmacyUniversity of Tasmania
  • Noel W. Davies
    • Central Science LaboratoryUniversity of Tasmania
  • Natasha L. Wiggins
    • School of Plant ScienceUniversity of Tasmania
Article

DOI: 10.1007/s10886-012-0188-5

Cite this article as:
McLean, S., Davies, N.W. & Wiggins, N.L. J Chem Ecol (2012) 38: 1318. doi:10.1007/s10886-012-0188-5

Abstract

The common brushtail possum (Trichosurus vulpecula) is the most widespread browsing marsupial in Australia, where it occupies woodland, agricultural, and urban environments. Following its introduction into New Zealand in the 19th century it has become a major feral pest, threatening native forests. The adaptability of the possum is thought to be due in part to its social organization, in which chemical communication is important. Possums have cloacal glands and exhibit related marking behavior. This study sought to characterize the chemicals involved in scent marking. Swabs were taken of the cloacal region of 15 possums (5 females, 10 males) from north-eastern Tasmania and analyzed by gas chromatography–mass spectrometry. There was a large number of compounds present, including 81 branched and unbranched, and saturated and unsaturated, fatty acids (C4–C15) and alcohols (C6–C26); 27 esters of 2,6- and 2,7-dimethyloctanol; 29 esters of formic acid; 39 sulfur compounds including S8 and a series of dialkyl disulfides, trisulfides, and tetrasulfides (C4–C10); and several alkylglycerol ethers. Many of these cloacal compounds are new to biology. There was considerable individual variability in the relative amounts of compounds found, and no evident sex differences, although the study was not designed to test this. This pattern suggests that these compounds may be acting collectively as a signature mixture of semiochemicals, carrying information on the individual, its kinship, and physiological and social status. This is the first detailed description of putative semiochemicals in any marsupial species.

Keywords

Mammalian chemical signalsBrushtail possumTrichosurus vulpeculaInvasive vertebrate pestCloacaFatty acidsFatty alcoholsDecyl estersFormate estersPolysulfidesAlkylglycerol ethers

Introduction

The brushtail possum (Trichosurus vulpecula) is the most widely-distributed marsupial in Australia, where it occupies a variety of habitats from the temperate forests of Tasmania to the arid center and the open tropical forests of the north (Kerle, 1984; Tyndale-Biscoe, 2005). Brushtail possums (‘possums’) are arboreal browsers that mature at 1 year and can weigh up to 5 kg in Tasmania but are smaller (about 2 kg) in northern Australia. Possums can subsist on almost any vegetation available, and with good nutrition can give birth to a single pouch young twice a year. Thus, under favourable conditions, the population growth can be rapid, leading to overbrowsing and destruction of vegetation. The brushtail possum has colonized farming and urban areas, and following its introduction into New Zealand in the 19th century its population has grown to densities considerably higher than in its native range (Green, 1984). In Australia, the possum is protected as an important species in native forest ecosystems; however, it is also a pest to plantation forests and agriculture, and large numbers are permitted to be culled annually to protect these industries (Coleman et al., 1997). In New Zealand, the possum has become a major vertebrate pest that threatens native forests (which are relatively unprotected by plant secondary metabolites) and birds, and acts as a vector for bovine TB (Cowan, 2001). In both countries, there are ecological and economic reasons for a better understanding of possum population dynamics and a need for more effective and humane management of pest populations.

The success of the possum under different environmental conditions may in part be due to its ability to alter its behavior and social organization (Kerle, 1984). Possums are nocturnal and, in Australia, solitary and spend less than 1 % of their time interacting directly, except during the breeding season when males have been observed to consort with a female for a number of days before mating (Day et al., 2000). This behavior may be different in New Zealand, where one study using data loggers has found that even in the breeding season contacts between males and females are infrequent (1 in 2 d) and mostly brief (<1 min) except when mating presumably occurred (Ji et al., 2005). When possums first meet, they establish their dominance relationship, and thereafter avoid encounters through individual recognition based on scent, as well as auditory and visual signals (Spurr and Jolly, 1999; Day et al., 2000). Their social structure involves dominance hierarchies, home territories (which can overlap) and dens, and behavioral contact usually is limited to the defense of individual space (Green, 1984; Day et al., 2000). A major factor that determines population densities may be the exclusion of juveniles and other subordinates from food resources and dens by the adult population. Dominance behavior also may limit breeding by subordinate males and females (Spurr and Jolly, 1999).

There exists behavioral and physiological evidence that the possum uses chemical communication, although no possum pheromones have been identified to date. Possums exhibit marking behavior in which they rub their chin, sternum, or cloaca on surfaces such as tree trunks and limbs (Biggins, 1984). The sternum often shows a distinctive wet patch, especially in sexually mature males, due to the presence of large and active apocrine and sebaceous glands (Bolliger and Hardy, 1944; Green, 1963). Analyses of sternal secretions have found a variety of compounds, including fatty acids, small heterocyclics, phenolics, and aldehydes (Salamon, 1995; Zabaras et al., 2005). The major components appear to be waxy lipids, possibly cholesterol esters of C16 and C18 fatty acids (Woolhouse et al., 1994).

There are two or more pairs of glands in the walls of the cloaca (Bolliger and Whitten, 1948; Green, 1963). One pair is large, ventral, and contains a thick, greasy, malodorous fluid: the oil secreting holocrine glands. The other glands consist of up to three pairs of small, dorsal, apocrine glands. Woolhouse et al. (1994) found that the holocrine glands contained triacyl glycerides and free low molecular weight (C7–C9) branched fatty acids. Saponification produced longer chain acids (C14, C16 and C18) from hydrolysis of the lipids. The apocrine glands contained regular triacyl glycerides, which on hydrolysis yielded the usual C10–C18 fatty acids.

This study aimed to identify potential semiochemicals in the cloacal secretions of brushtail possums. The cloaca was chosen because the presence of cloacal glands suggests that they may have an important secretory function. Secretions may be altered by the action of bacteria (Alberts, 1992), and we considered that swabs of the cloacal surface would be most representative of the chemicals deposited on a marked site. Our analysis of cloacal swabs has identified a large number of compounds in several chemical groups: free fatty acids and alcohols, esters of fatty acids and branched decanols, esters of formic acid and fatty alcohols, and sulphur compounds. This paper describes the identification of these putative semiochemicals and their relative amounts, and discusses their likely biological significance. It is the first detailed description of putative semiochemicals in any marsupial species.

Methods and Materials

Animals

Samples were taken from possums culled under agricultural permit on a grazing property in north-eastern Tasmania on one night in November (Tasmanian Parks and Wildlife Permits 2803362, 2801251). Swabs were taken within 30 min of death. The group comprised five females (all adults) and ten males (9 adults and 1 juvenile).

Sample Collection

Swabs were taken with binder-free glass microfibre filter paper (25 mm GFC, Whatman, England). To avoid contamination, filters were placed on clean office paper and handled with forceps. The cloacal target region comprised the edges and inside of the cloaca, as well as its characteristic long hairs, and was wiped several times with the swab. The swabs were then placed into a glass vial, sealed with a teflon-lined screw cap, and stored at −20 °C.

Chemical Analysis

A subsample of the filter was cut off for analysis, usually approximately 5x5 mm. Initial experiments indicated that the analysis by gas chromatography–mass spectrometry (GC-MS) had to be capable of measuring components that varied greatly in volatility and polarity as well as in the amounts present in the sample. Thermal desorption (TD) analysis was first used, as it entailed least interference with the sample and avoided a solvent peak that would interfere with early-eluting compounds. Thermal desorption analysis was used for sulphur compounds and formate esters. However, polar compounds chromatographed poorly, and the less-volatile compounds were not efficiently introduced onto the column at the 150 °C used for desorption. This was overcome by using solvent extraction and split/splitless injection. Usually, the extract was treated to form trimethylsilyl (TMS) derivatives, as this gave better chromatography of polar components, but the underivatized data were useful for identification of some compounds. Once it was realized that many of the compounds were esters, some samples were treated with base to hydrolyze the esters to the free acids and alcohols, thus assisting in their analysis. Split/splitless injection was used for acids, alcohols, and decyl esters.

The gas chromatograph was a Varian CP-3800 with CP-8400 autosampler coupled to a Varian 1200 L triple quadrupole mass spectrometer. Injections of 1 μl were by a Varian 1177 split/splitless injector at 250 °C. The GC column was a 30 m × 0.25 mm capillary column with a 5 μm coating of 5 % phenyl methyl silicone (Factor Four VF-5MS, Varian, Inc.). GC operating conditions were: He 1.2 ml/min, temperature 50 °C for 2 min, then 8 °C/min to 300 °C and held for 5 min (run time 38.25 min). EI mass spectra were recorded in full scan mode. MS operating conditions were: source temperature 220 °C, transfer line 290 °C, ionizing energy −70 eV, scan range m/z 35–600, scan time 0.31 sec.

For solvent extraction, a piece of filter was placed in a glass vial with teflon-lined cap and 0.25 ml chloroform (HPLC grade, Omniscribe) were added. The vial stood overnight in a 4 °C refrigerator, then an aliquot was placed in a 150 μl insert in a teflon-capped autosampler vial for split (or splitless) injection. For preparation of TMS derivatives, a 50 μl aliquot of the chloroform extract was mixed with an equal volume of TMS reagent (trimethylsilyltrifluoroacetamide with 1 % trimethylchlorosilane, Sigma-Aldrich®) in an autosampler vial and stood at 60 °C for 1 hr. To hydrolyze esters, a piece of filter was placed in a 10 ml glass vial with 400 μl 2 M KOH in 50 % ethanol, capped, and heated at 80 °C for 1 hr, then stood at room temperature for 24 hr. The hydrolyzate was acidified with 2 M HCl, extracted with ethyl acetate (2 × 0.5 ml), taken to dryness on a rotary evaporator, and redissolved in 200 μl chloroform and kept at −20 °C until analysis. Dimethyl disulfide (DMDS) diadducts of unsaturated formate esters were prepared as described by Carlson et al. (1989).

For TD analysis, a piece of the filter paper was inserted into a stainless steel tube (89 mm × 6.4 mm O.D.), inactivated by a very thin layer of quartz bonded to the inside surface (Silicosteel, Markes International, UK). This sample tube was placed in a TD unit that incorporated a cold trap (Unity Thermal Desorption Unit, Markes Int., UK) and was connected in turn to the GC-MS system described above. The cold trap contained a carbon molecular sieve (U-T6SUL, Markes Int., UK) and was operated at 25 °C, while the connecting He gas lines were kept at 200 °C. Analysis was begun by heating the sample tube to 150 °C for 10 min which mobilized volatile organic compounds that were then collected in the trap. Chromatography was initiated by reversing the flow through the trap on to the GC column, and rapidly (in 3 sec) heating the trap to 290 °C. This temperature was maintained for 3 min, which removed all of the volatile material and prepared the trap for the next analysis. The GC operating conditions were: He 0.5 ml/min, oven temperature program 40 °C for 2 min, then 8 °C/min to 290 °C and held for 2 min (run time 35.25 min).

The reproducibility of subsampling from the filters was examined by analysis of a second cutting on another day. Using swabs from eight animals, and the areas of 27 peaks in a TD chromatogram, the median correlation between the two analyses was r2 0.927 (range 0.749–0.948).

Reference compounds were purchased from Sigma-Aldrich® and are labelled in the Tables. Formate esters were synthesized from the alcohols by heating in excess 100 % formic acid (Burger et al., 1996). Kovats indices (KI) were determined by the analysis of a series of standard hydrocarbons on the same day as the possum samples were analyzed.

Search Strategy

Chromatograms of cloacal samples showed a large number of peaks, many of which contained more than one component. Initial identifications were made by matches in the NIST mass spectral data base (National Institute of Standards and Technology Mass Spectral Search Program Version 2.0f (2008), NIST/EPA/NIH Mass Spectral Library) together with KI values from the NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/). Other compounds were provisionally identified by first principles interpretation of their MS and KI data. Where possible, reference compounds were purchased or synthesized to confirm identifications. For most compounds, identification was assisted by their relationship to a group of isomers or homologues, which were revealed with ion chromatograms. Additional data came from chemical reactivity: hydrolysis of esters and DMDS adduct formation by unsaturated compounds.

Sulfur compounds and formate esters were introduced to the GC by TD, and acids and alcohols (as their TMS derivatives) and decyl esters by split/splitless injection. For each chemical group, an automated search method was developed with the instrument software, using retention time (± 0.2 min) and the quantitative ion (QI) or a reference spectrum to identify the target compounds. The automated reports on each sample were checked manually and corrected for false negatives (e.g., if the retention time fell just outside the time window), or false positives (e.g., if the QI was unsupported by other identifying ions).

Quantitation

Since reference compounds were unavailable, the relative amount of each compound was estimated from the ratio of its peak area (measured by the total ion current, TIC) to the total peak areas for all compounds in the same chemical group. Each group was considered separately since MS detector sensitivity differs for different chemical compounds but can be remarkably similar within a chemical group (Göröcs et al., 2012). It was considered preferable to give this indication of the relative amounts (%) of compounds within each group (which clearly differed greatly), rather than to omit the data. Comparisons between animals were unaffected by this consideration, since the relationship between TIC and amount of a particular compound was the same for each sample.

The chromatograms were complex and many of the peaks comprised more than one compound. Therefore, the TIC was calculated from the peak area of a single quantitative ion (QI), usually the highest significant mass unless there was interference from another compound. For each compound, a good quality mass spectrum was chosen to calculate the ratio of the abundance of the QI to the TIC. Measuring a single QI avoided chromatographic interference, and enabled the TIC to be calculated for each compound by dividing the QI peak area by the QI/TIC ratio. For several analytes, it was not possible to obtain a mass spectrum sufficiently free from interfering compounds to calculate the QI/TIC ratio, and a value was assigned from either a structural isomer, the average of the two adjacent homologues or the value of a homologue. In some other cases the QI/TIC ratio was calculated despite a contaminated mass spectrum which would result in an underestimated value.

Results

Group 1: Acids and Alcohols

Initial Identification

GC-MS analysis of cloacal swabs of the common brushtail possum gave complex chromatograms that included esters and their free fatty acids and alcohols. As reported by Woolhouse et al. (1994), there were large amounts of neutral lipids, principally as triacyl glycerides, and these were not further investigated. Hydrolysis of the esters and TMS derivatization facilitated the identification of the large number of fatty acids and alcohols (Table 1). The TMS derivatives of Cn acids and Cn+1 alcohols produced an [M-CH3]+ ion of the same mass, enabling a convenient search for these compounds with ion chromatograms such as those shown in Fig. 1. For example, the chromatogram of m/z 201 showed peaks for four C8 acids and one C9 alcohol, which were confirmed by their full mass spectra. Although there also were peaks for an unknown compound (U), a C9 acid (19) and C10 acid (20), these were distinguishable by their mass spectra and, for the homologous acids, their larger peaks in other ion chromatograms (in this case, m/z 215 and 229, respectively). Note that compound 9, 2-methylhexanoic acid, was seen only in the TIC chromatogram as it did not give a strong m/z 187 peak.
Table 1

Acids and alcohols found in brushtail possum cloacal swabs, as TMS derivatives

No.

Chemical Class

Compound

LEa

MW (TMS)

KI Found

KIb Lit.

QId (m/z)

QI/TICe

1

C4 acid

n-Butanoic acid

A

160

892

889R

145

0.232

2

C6 alcohol

2-Methyl-3-pentanol

B

174

923

918

131

0.191

3

C6 alcohol

2-Hexanol

B

174

925

919

117

0.394

4

C6 acid

4-Methylpentanoic acid

B

188

1037

1040

173

0.142

5

C7 alcohol

3-Methylhexanol

B

188

1044

1042

173

0.126

C3 hydroxyacid

2-Hydroxypropanoic (lactic) acid

A

234

1054

1053R

191

0.054

6

C6 acid

n-Hexanoic acid

B

188

1070

1069

173

0.074

7

C7 acid

5-Methylhexanoic acid (iso)

C

202

1130

 

187

0.117

8

C7 acid

4-Methylhexanoic acid (anteiso)

C

202

1138

 

187

0.123

9

C7 acid

2-Methylhexanoic

C

202

1158

 

145

0.154

10

C7 acid

n-Heptanoic acid

A

202

1167

1166R

187

0.166

C10:1 alcohol

Citronellol

C

228

1210

 

214

0.021

11

C8 acid

6-Methylheptanoic acid (iso)

C

216

1222

 

201

0.105

12

C8 acid

5-Methylheptanoic acid (anteiso)

C

216

1227

 

201

0.110

13

C8 acid

Octanoic acid 3

D

216

1232

 

201

0.065

14

C9 alcohol

Nonanol branched

A

216

1239

 

201

0.111

C7 acid

Benzoic acid

A

194

1246

1247R

194

0.017

15

C8 acid

n-Octanoic acid

A

216

1262

1262R

201

0.133

C3 triol

Glycerol

B

308

1268

1266

205

0.092

16

C10 alcohol

2,7-Dimethyloctanol

B

230

1272

 

215

0.128

17

C10 alcohol

2,6-Dimethyloctanol

C

230

1276

 

215

0.081

C4 acid

Succinic acid

B

262

1312

1314

247

c

18

C9 acid

7-Methyloctanoic acid (iso)

C

230

1322

 

215

0.123

19

C9 acid

6-Methyloctanoic acid (anteiso)

C

230

1328

 

215

0.114

20

C10 acid

2,7-Dimethyloctanoic acid

C

244

1334

 

229

0.090

21

C10 acid

2,6-Dimethyloctanoic acid

C

244

1339

 

229

0.073

C10 alcohol

9-Hydroxycineole

A

242

1347

1348R

139

0.235

22

C9 acid

n-Nonanoic acid

A

230

1358

1359R

215

0.143

23

C10 acid

Decanoic acid, branched

D

244

1365

 

229

0.075

Phenol

Resorcinol

A

254

1378

1379R

254

0.115

C10 acid

Citronellic acid

B

242

1385

1383

227

0.007

C10 acid

Myrtenoic acid

B

238

1409

1411

223

0.029

C10 acid

9-Cineolic acid

C

256

1411

 

139

0.235

C9 acid

Benzene propanoic acid

B

222

1417

1414

222

0.025

C8 acid

Salicyl alcohol

A

268

1429

1430R

268

0.041

Phenol

Orcinol

A

268

1443

1443R

268

0.104

24

C10 acid

n-Decanoic acid

A

244

1454

1456R

229

0.106

25

C11 acid

9-Methyldecanoic acid (iso)

C

258

1516

 

243

0.069

26

C11 acid

8-Methyldecanoic acid (anteiso)

C

258

1522

 

243

0.077

27

C11 acid

n-Undecanoic acid

A

258

1552

1553R

243

0.098

28

C12 acid

Dodecanoic acid 1

D

272

1558

 

257

0.034

29

C12:1 acid

Dodecenoic acid 1 (Cis-5-)c

B

270

1637

1631

255

0.019

30

C12:1 acid

Dodecenoic acid 2

D

270

1644

 

255

0.039

31

C12 acid

n-Dodecanoic acid

A

272

1650

1650R

257

0.067

32

C13 alcohol

n-Tridecanol

A

272

1663

1663R

257

0.140

33

C13 acid

Tridecanoic acid 1

D

286

1691

 

271

0.031

34

C13 acid

Tridecanoic acid 2

D

286

1719

 

271

0.029

35

C13 acid

n-Tridecanoic acid

A

286

1748

1747R

271

0.051

C9 hydroxyacid

Hydrocinnamic acid

B

310

1758

 

310

0.055

36

C14 alcohol

n-Tetradecanol

A

286

1761

1760R

271

0.112

C9 diacid

Nonadioic acid

B

332

1793

1788

317

0.033

37

C14:1 acid

Tetradecenoic acid 1 (Cis-6-)c

C

298

1824

1819

283

0.012

38

C14:1 acid

Tetradecenoic acid 2

D

298

1834

 

283

0.026

39

C14 acid

n-Tetradecanoic acid

A

300

1846

1845R

285

0.033

40

C15 alcohol

n-Pentadecanol

A

300

1859

1858R

285

0.071

41

C15 acid

Pentadecanoic acid 1

D

314

1884

 

299

0.012

42

C15 acid

Pentadecanoic acid 2

D

314

1908

 

299

0.026

43

C15 acid

Pentadecanoic acid 3

D

314

1916

 

299

0.029

44

C15:1 acid

Pentadecenoic acid

D

312

1921

 

297

0.009

45

C15 acid

n-Pentadecanoic acid

A

314

1944

1944R

299

0.023

46

C16 alcohol

n-Hexadecanol

A

314

1956

1955R

299

0.076

47

C16:1 acid

Hexadecenoic acid 1

D

326

2018

 

311

0.028

48

C16:1 acid

Hexadecenoic acid 2 (Cis-9-)

A

326

2022

2023R

311

0.066

49

C16:1 acid

Hexadecenoic acid 3

D

326

2033

 

311

0.067

50

C16 acid

n-Hexadecanoic acid

A

328

2043

2043R

313

0.115

51

C17 alcohol

n-Heptadecanol

A

328

2055

2054R

313

0.147

52

C17 acid

Heptadecanoic acid 1 (8-Me-C16)cc

C

342

2080

2081

327

0.051

53

C17:1 acid

Heptadecenoic acid 1

D

340

2087

 

325

0.023

54

C17:1 acid

Heptadecenoic acid 2

D

340

2116

 

325

0.043

55

C17:1 acid

Heptadecenoic acid 3 (Cis-10-)c

B

340

2127

2126

325

0.036

56

C17 acid

n-Heptadecanoic acid

A

342

2142

2141R

327

0.081

57

C18 alcohol

n-Octadecanol

A

342

2153

2152R

327

0.170

58

C18:2 acid

Octadecadienoic acid 1

D

352

2197

 

337

0.006

59

C18:2 acid

Octadecadienoic acid 2

D

352

2203

 

337

0.031

60

C18:1 acid

Octadecenoic acid 1 (Cis 9-)

A

354

2214

2214R

339

0.036

61

C18:1 acid

Octadecenoic acid 2 (Trans-11-)c

B

354

2221

2223

339

0.058

62

C18:1 acid

Octadecenoic acid 3 (Cis-13-)c

B

354

2232

2232

339

0.041

63

C18 acid

n-Octadecanoic acid

A

356

2241

2241R

341

0.063

64

C19 alcohol

n-Nonadecanol

B

356

2251

2246

341

0.147

65

C19 acid

n-Nonadecanoic acid

B

370

2339

2338

355

0.059

66

C20 alcohol

n-Eicosanol

B

370

2350

2351

355

0.110

67

C20:2 acid

Eicosadienoic acid 1

D

380

2394

 

365

0.019

68

C20:2 acid

Eicosadienoic acid 2

B

380

2402

2413

365

0.027

69

C20:1 acid

Eicosenoic acid 1

D

382

2411

 

367

0.040

70

C20:1 acid

Eicosenoic acid 2 (Cis-11-)c

B

382

2421

2420

367

0.034

71

C20:1 acid

Eicosenoic acid 3 (Cis-13-)c

B

382

2431

2426

367

0.021

72

C20 acid

n-Eicosanoic acid

B

384

2439

2447

369

0.055

73

C21 alcohol

Heneicosanol

B

384

2448

2445

369

0.114

74

C21 acid

Heneicosanoic acid

B

398

2537

2534

383

0.052

75

C22 acid

Docosanoic acid

B

412

2636

2632

397

0.052

76

C23 alcohol

Tricosanol

B

412

2645

2640

397

0.114

77

C23 acid

Tricosanoic acid

B

426

2736

2732

411

0.052

78

C24 alcohol

Tetracosanol

B

426

2743

2739

411

0.114

79

C24 acid

Tetracosanoic acid

B

440

2834

2829

425

0.052

80

C25 acid

Pentacosanoic acid

B

454

2934

2931

439

0.052

81

C26 alcohol

Hexacosanol

B

454

2941

2936

439

0.114

Steroid

Cholesterol

B

458

3157

3150

458

c

aLevels of evidence: A, matched reference standard; B, matched literature values MS and KI; C, interpretation of MS and KI; D, MS and KI indicate chemical class

bLiterature value or reference standard (R)

cPossible isomer, based on KI match

dQI is the quantitative ion used to determine the relative amount of each compound

eQI/TIC is the ratio of the peak area of the QI to the total ion current of all ions for that compound

https://static-content.springer.com/image/art%3A10.1007%2Fs10886-012-0188-5/MediaObjects/10886_2012_188_Fig1_HTML.gif
Fig. 1

Chromatogram showing peaks for acids and alcohols 7–24 in Table 1. The cloacal sample, from a male possum, was hydrolyzed and derivatized with TMS reagent. The top panel shows the total ion current (TIC) and the other panels show chromatograms of [M-15]+ ions for acids from C7 (m/z 187) to C10 (m/z 229), and C9 and C10 alcohols (m/z 201 and 215). A, acetic acid; B, benzoic acid; C, citronellol; CA, citronellic acid; U, unknown

The homologous series of n-acids from C6 to C25 were identified as their TMS derivatives and, as expected, their KI values showed an increase of approximately 100 units for each additional carbon. Linear regression analysis of KI vs. carbon number gave r2 = 1.000, slope = 98.2 ± 0.1, P < 0.001, df 1, 18. The same chromatograms showed the series of n-alcohols from C13 to C26. Linear regression analysis of KI vs. carbon number gave r2 = 1.000, slope 98.3 ± 0.1, P < 0.001, df 1, 10. For normal isomers, the Cn acid eluted about 13 KI units before the Cn+1 alcohol (Table 1). The acids were further characterized by the ion m/z 117, while alcohols had an even stronger [M-15]+ ion (often 100 %) and an ion at m/z 103. The mass spectra and KI values of the n-acids and alcohols were in good agreement with published data (NIST Chemistry WebBook) and the available standards (Table 1).

Branched isomers eluted earlier than their corresponding normal isomers, as shown for the acids (e.g., 7, 8) and early-eluting C10 alcohols (16, 17) in Fig. 1. Unsaturation also resulted in earlier elution, but as the mass also decreased an additional ion chromatogram was required to show these isomers. Figure 2 shows the saturated and unsaturated C18 acids and a C19 alcohol. There were some unresolved nonadecenoic acids (N) that were not quantitated. There were also some alkylglycerol ethers (G) that remained after hydrolysis had removed the acyl groups from their lipid precursors. These compounds are described in Group 5.
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Fig. 2

Another part of the chromatogram in Fig. 1 showing peaks for acids and alcohols 47–74, and ion chromatograms showing saturated and unsaturated C18 acids and a C19 alcohol. G, glycerol ethers; N, nonadecenoic acids (not further identified)

The identification of particular groups of acids and alcohols is described below. All compounds are listed in Table 1, together with the level of evidence used for their identification. Compounds that were readily identified from their MS and KI data are not discussed. Table 1 also lists 15 common urinary metabolites of the brushtail possum (the compounds not numbered), most of which are diet related phenols or terpene metabolites. As these are unlikely to be products of the paracloacal gland secretions, they have not been further considered here. Additional mass spectral data on compounds 2–5 are given in Supplementary Table 1.

Iso and Anteiso Acids

In Figure 1, the ion chromatograms for the C7, C8 and C9 (but not C10) acids each showed a pair of compounds that were characterized, respectively, by a methyl substituent at the ω-2 (iso) and ω-3 (anteiso) positions. These iso and anteiso acids were first found in possum paracloacal glands by Woolhouse et al. (1994), who confirmed their structures by synthesis of standards. We can now further characterize the compounds and two analogous C11 acids with mass spectral and retention time data. (Table 1 and Supplementary Table 2). The TMS derivatives all showed a major [M-15]+ ion and a strong ion at m/z 117 indicative of a carboxylate (COOTMS) fragment. The iso isomers (7, 11, 18, 25), which eluted first, showed a more prominent [M-43]+ ion, indicating loss of C3H7, while for the anteiso isomers (8, 12, 19, 26) the loss of 29 (C2H5) and in most cases 57 (C4H9) were relatively greater. The same pattern of losses was even more pronounced with the underivatized C7 and C9 acids (the C8 acids were less abundant and not detected in underivatized form). The underivatized MS of compound 8 matched the NIST spectrum for 4-methylhexanoic acid. The shift in retention indices of the TMS derivatives provided additional evidence that the branching was similar in these pairs of isomers. The iso isomers eluted 36–40 Kovats units before the corresponding n-acids and the anteiso isomers followed 5–8 units later (Table 1).

C10 Alcohols and Acids

Mass spectral data for C10 alcohols and acids are given in Supplementary Table 3. The ion chromatogram for m/z 215 (Fig. 1) shows two branched C10 alcohols (16 and 17) that eluted before the C9 acids. Analysis of the underivatized alcohols showed that the mass spectrum of 16 matched 2,7-dimethyloctanol (NIST replicate MS). Underivatized 17 had a similar MS except that m/z 111 (possibly a loss of 18 + ethyl) was much stronger and m/z 97, consistent with the loss of propyl, was weaker. The TMS derivatives of both compounds showed a strong m/z 103 ion, consistent with the formation of [CH2OTMS]+, facilitated by alpha-cleavage to both the 2-methyl group and the terminal carbon bearing the hydroxyl group. We propose that compound 17 is 2,6-dimethyloctanol.

The chromatogram of m/z 229 (Fig. 1) showed n-decanoic acid (24) and three branched isomers eluting earlier. The acids 20 and 21 had similar peak profiles and retention differences to the branched decanols 16 and 17, suggesting that they may be the corresponding acids (2,7- and 2,6-dimethyloctanoic acid). Both compounds showed an abundant ion at m/z 146 (which can arise from McLafferty rearrangement of a 2-methylcarboxylate TMS group to yield [CH3CH=C(OH)-OTMS]+.), indicating a 2-methyl substituted carboxylic acid. When underivatized, acids 20 and 21 underwent a similar rearrangement to give a base peak at m/z 74 ([CH3CH=C(OH)2]+.), supporting the 2-methyl substituent. Acid 20 showed an ion at m/z 129 corresponding to a loss of 43 (C3H7), and this was absent in 21 that, however, showed a loss of 57 (C4H9) corresponding to a branched butyl group.

Other Branched and Unsaturated Isomers

Using the unbranched compounds as reference points, branched isomers were found from peaks of the same mass that eluted earlier, as described above for the iso and anteiso acids. However, the detailed structures were not determined for the remaining branched compounds. For example, dodecanoic acid 1 (28) is an unknown isomer that eluted 92 KI units before n-dodecanoic acid (31), and is probably doubly-branched because of this large shift in KI.

Similarly, unsaturated isomers were found by plots of their [M-15]+ ion which, for each double bond, is two mass units lower than for the saturated compound. Although individual ion chromatograms were not completely specific for the isomers of interest, there was no confusion once each peak’s mass spectrum was viewed. For example, the C18:1 acids, 60 and 61, gave peaks on the m/z 341 (C18:0) plot, but their full mass spectra showed that this ion was just an isotope of the m/z 339 ion. Compound 60 matched standard cis-9-octadecenoic (oleic) acid, but neither 58 or 59 matched standard linoleic acid (KI 2209). Similarly, 48 was cis-9-hexadecenoic (palmitoleic) acid. Several isomeric C19:1 acids were evident (N in Fig. 2), but as they did not resolve they were not characterized.

It was not possible to assign unique structures to the other compounds, which are described in Table 1 by their compound class. In some cases, an isomer has been suggested based on a literature KI value. Supplementary Table 4 gives GC-MS data on the underivatized acids and alcohols from C12 to C18.

Quantitation of Acids and Alcohols

Although the acids and alcohols were, for analytical reasons, identified in hydrolyzed samples, all were present in the free form in unhydrolyzed samples, and these were used for quantitation since this was the biologically relevant form. The median number of compounds found per animal was 56 (range 16–71), and there was no sex difference. The possum with fewest compounds was a juvenile male, but its mother also had few compounds (23), suggesting that the number of compounds produced is not simply determined by maturity. Similarly, the relative amounts varied greatly although this was also a function of the number of compounds found. Table 2 shows the relative amounts of compounds that comprised >1 % of the total acids plus alcohols in males or females. Individual amounts varied greatly, as shown by the large SD values, and there was no significant sex difference, although the study was not designed to test this.
Table 2

Acids and alcohols with relative amountsa greater than 1% in either male or female possums

Compound No.a

Relative amount (%)b

Females (N = 5)

Males (N = 10)

Mean

SD

Meanc

SD

2

27.98

30.18

22.33

23.62

3

13.70

14.46

11.26

11.80

7

7.34

8.65

7.57

9.19

8

10.45

11.22

9.02

6.84

12

2.83

5.36

0.59

0.65

16

3.23

6.71

0.98

1.52

18

3.37

3.69

1.71

1.30

19

6.62

8.60

3.23

2.62

31

1.18

1.73

0.88

0.69

37

0.31

0.46

1.24

1.13

38

1.04

1.03

2.18

1.89

39

3.89

4.56

6.41

4.89

40

2.26

1.96

4.45

2.79

44

0.23

0.46

0.95

1.18

45

1.58

1.87

5.51

6.45

47

0.79

0.64

2.61

2.53

50

2.37

2.44

3.71

2.20

59

0.78

0.70

2.13

1.62

60

1.76

1.17

3.07

2.13

aCompounds are listed in Table 1

bRelative amount is the proportion of all 81 acids and alcohols listed in Table 1, calculated by the ratio of the TIC to the sum of all TICs

cThe means were not significantly different between females and males by unpaired t-test (P > 0.05)

In addition to biological variability, the swabs suffered a selective loss of the more volatile (early-eluting) components every time the sample tube was opened to take another subsample, which resulted in a relative decrease in the percentage of more volatile components and a relative increase in the percentage of less volatile (late-eluting) components. This change was noticed in the laboratory, but presumably also occurs on the animal and the marked substrates. Many compounds were present at very low percentages (36 at <0.1 %) but were nevertheless detectable. It was considered important to quantitate as many compounds as possible, since quantitatively minor compounds can act as pheromones (Walker et al., 2009).

Group 2: Decyl Esters

Initial Identification

Many of the chromatogram peaks were removed by hydrolysis, suggesting that they were due to esters. The two branched decyl alcohols (2,6- and 2,7-dimethyloctanol) described above (compounds 16 and 17 in Table 1) were found subsequently to be present mostly as a series of esters with acids from C7 to C20. Figure 3 shows a portion of a chromatogram that is dominated by the presence of decyl esters. These decyl esters were readily detected by characteristic alcohol-related ions, [C10H21]+ at m/z 141 and its olefinic rearrangement ion [C10H20]+. at m/z 140 (Beynon et al., 1968), which was invariably more prominent (Supplementary Table 5). Other decanol-related ions were [C10H21O]+ and [C10H21OCO]+ at m/z 157 and 185, respectively. There was also an ion at m/z 111, corresponding to the loss of an ethyl radical from the alkene [C10H20]+.. This ion was more prominent for 2,6-dimethyloctanol (51 %) than for its 2,7-isomer (4 %)(Supplementary Table 3). For the decyl esters, the m/z 111 ion was much stronger for decyl heptanoate 2 (100 %) than for decyl heptanoate 1 (12 %), suggesting that they are esters of 2,6- and 2,7-dimethyloctanol, respectively. Similar patterns were observed with all the other isomeric pairs of esters, except for C20:1, indicating that the 2,7-dimethyloctyl esters elute before their 2,6-isomers. This elution order in which the ω-2 branched isomer (iso) precedes the ω-3 (anteiso) isomer occurs generally, and was seen with the parent dimethyloctanols and four pairs of iso and anteiso acids (Table 1).
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Fig. 3

Chromatogram showing peaks for decyl esters 11–21 in Table 3. The cloacal sample was from a female possum. The top panel shows the total ion current (TIC), and the other panels show chromatograms of ions characteristic for the components of the decyl esters: C10 alcohols, and acids from C14–C16

Acyl Moieties

Loss of the olefinic fragment from an ester leaves a residue that corresponds to the acid moiety [R′COOH]+. or to an ion one unit higher [R′COOH2]+ (Beynon et al., 1968). These ions, especially the second, were used to confirm the acyl portions of the saturated esters shown in Table 3 and Supplementary Table 5. However, these ions were less abundant in unsaturated acids, which were instead characterized by ions which corresponded to [R′COOH-H2O]+. and [R′CO]+. The ion [R′]+ was usually not significant, and was not useful for identification.
Table 3

Decyl esters found in possum cloacal swabs. The decyl alcohols were 2,6- and 2,7-dimethyloctanol

Compound

KI

QIa

QI/TICa (m/z)

Relative amount (%)b

No.

Acyl moiety

Meanc

SD

1

Heptanoate 1d

1740

131

0.0657

0.97

2.07

2

Heptanoate 2d

1744

111

0.1130

2.34

4.48

3

Heptanoate 3

1749

131

0.0493

1.38

2.83

4

Nonanoate 1

1937

159

0.0868

0.54

0.83

5

Nonanoate 2

1943

159

0.0672

1.74

3.61

6

Undecanoate

2176

140

0.0225

0.03

0.10

7

Dodecanoate 1d

2277

140

0.0559

0.88

1.57

8

Dodecanoate 2d

2285

140

0.0248

0.09

0.21

9

Tridecanoate 1d

2378

140

0.0537

0.51

0.58

10

Tridecanoate 2d

2386

111

0.0323

0.03

0.08

11

Tetradecenoate 1d

2458

140

0.0238

1.27

2.03

12

Tetradecenoate 2d

2472

140

0.0384

4.31

3.43

13

Tetradecanoate 1

2480

140

0.0776

5.52

3.41

14

Tetradecanoate 2

2487

111

0.1170

3.79

4.82

15

Pentadecenoatec

2556

140

0.0338

1.51

1.58

16

Pentadecanoate

2581

140

0.0929

2.40

1.96

17

Hexadecadienoated

2643

140

0.0127

0.18

0.53

18

Hexadecenoate 1d

2653

140

0.0209

7.78

5.70

19

Hexadecenoate 2d

2672

140

0.0183

14.01

15.74

20

Hexadecanoate 1

2679

140

0.0426

16.72

5.07

21

Hexadecanoate 2d

2686

140

0.0163

5.48

7.21

22

Octadecadienoated

2837

279

0.0274

14.55

9.58

23

Octadecenoate 1e

2846

140

0.0454

9.06

3.93

24

Octadecenoate 2e

2855

140

0.0513

3.90

4.40

25

Octadecenoate 3d

2864

140

0.0459

0.79

1.56

26

Eicosenoate 1d

3025

140

0.0551

0.17

0.35

27

Eicosenoate 2d

3035

140

0.0214

0.05

0.16

aQI and QI/TIC are defined in Table 1 footnotes

bRelative amount is the proportion of all 27 decyl esters, calculated by the ratio of the TIC to the sum of all TICs

cMean and SD value for two females and seven males. The remaining animals had no decyl esters

dPeak contained more than one isomer

eThese adjacent peaks were not fully resolved, and may also contain additional isomers

The elution order supported the ester empirical formulae, increasing with mass and with the unsaturated isomers eluting earlier (Fig. 3). The acid portion of the esters could only be characterized by carbon number and degree of unsaturation, while the position of any branching and double bonds could not be determined from the mass spectral data alone. Additionally, some of the chromatogram peaks were broad and showed evidence of splitting for some compounds, indicating that more than one isomer was present (Table 3). This was not unexpected, since we had found many isomers of fatty acids in the samples (Table 1). For example, there were four heptanoic acids, two of which (the iso and anteiso isomers) were major components, and these with the two decanol isomers could form four decyl heptanoate esters, and three were found. There were also three octadecenoic acids, potentially producing six decyl octadecenoates, which could account for the very broad peaks found for these compounds, with at least two partially-resolved isomers. No decyl esters were found for C8, C10, C17, C19, C20:0, and C21–23 acids, although these acids had been found in the cloacal samples. Conversely, there was good evidence for decyl hexadecadienoate, although the C16:2 acid was not found.

Although in general it was not possible to relate the decyl esters to specific acids found previously, some patterns emerged. Previously, we found only the n-isomers of tetradecanoic and hexadecanoic acid, indicating that the two decyl esters found for these acids are, in order, 2,7- and 2,6-dimethyloctyl tetradecanoate and hexadecanoate. However, the other saturated acids had branched isomers, which in some cases were more abundant than the n-isomer (e.g., C7 acids in Fig. 1). This branching also would affect retention indices, thus confounding any analysis based on KI. It may be that the acyl groups in decyl esters reflect the relative amounts of the acid isomers. The n-acyl isomers are expected to elute last, and this was supported by the linear regression of KI vs. carbon number for these isomers (r2 0.9991, slope 105.0 ± 1.3, P < 0.001, df 1, 6).

Quantitation of Decyl Esters

The median number of decyl esters found per animal was 7 (range 0–27), with no sex difference. The relative amounts of decyl esters are shown in Table 3. No decyl esters were found in three females and three males, and these samples were excluded from the group analysis. With only two females remaining and no obvious sex differences, the female and male data were combined. The most abundant compounds were the C16:0, C16:1, and C18:2 esters, with other C16, C18, and C14 esters also significant. The corresponding acids were among the more abundant acids in Table 1. As indicated by the large SD values, there was considerable individual variability in the relative amounts of the decyl esters.

The six animals which had no decyl esters mostly had undetectable levels of the precursor 2,7- and 2,6-dimethyloctanols. For these siz animals, the median (range) relative amounts for the alcohols were, respectively, 0.01 % (0–0.06) and 0.01 % (0–0.05), whereas the values for the nine animals with decyl esters present were 0.95 % (0.08–15.21) and 0.21 % (0.06–3.46). The differences were highly significant for both alcohols (Mann–Whitney U test, P < 0.001). Similarly, there were zero levels of the corresponding dimethyloctanoic acids (compounds 20 and 21 in Table 1) in the animals without decyl esters, and significant (non-zero by the Wilcoxon Signed Rank Test) levels in the animals with decyl esters (compound 20: 0.10 (0–0.055) P = 0.016; compound 21: 0.01 (0–0.11) P = 0.031).

Group 3: Formate Esters

Initial Identification

Chromatograms of the TD analyses of cloacal samples showed a region (18–26 min; KI 1635-2243) that was dominated by the peaks of esters of formic acid with fatty alcohols (Fig. 4). These peaks were not at first identified, as their major ions were merely typical of many aliphatic compounds, but their disappearance after hydrolysis and failure to form TMS derivatives indicated that they were esters. Table 4 shows the KI and MS data for the formate esters found (see also Supplementary Table 6). The literature KI values were calculated using the retention time data of Garcia-Rubio et al. (2002). All MS showed the characteristic formate ion [HCOOH2]+ at m/z 47, although this was of low abundance (<2 %). There was a related ion corresponding to the loss of HCOOH at [M-46]+., as well as an ion at [M-74]+. corresponding to the additional loss of C2H4. These ions have been reported previously in the EI mass spectra of formate esters (Mo et al., 1995; Burger et al., 1996). The [M-18]+. ion was generally small or absent. The relative abundance of these ions was affected by the degree of unsaturation, with the largest of the identifying ions being due to [M-74]+. for alkanyl formates, [M-46]+. for alkenyl formates, and M+. for alkadienyl formates. These ions were used to search for formate esters using ion chromatograms (as shown in Fig. 4 for formate esters of alcohols from C16–C18) and were mostly used as the QI (Table 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs10886-012-0188-5/MediaObjects/10886_2012_188_Fig4_HTML.gif
Fig. 4

Chromatogram showing peaks for formate esters 8–29 in Table 4. The cloacal sample was from a male possum. The top panel shows the total ion current (TIC), and the other panels show ions characteristic for formate esters (m/z 47) and for saturated and unsaturated formates of alcohols from C16:1 to C18:0. The C19 formate esters (24–29) are only shown in the TIC and m/z 47 chromatograms

Table 4

Formate esters found in possum cloacal swabs

Compound

LEa

KI

QIb (m/z)

QI/TICb

Relative amount (%)c

No.

Type

Alcohol moiety

Meand

SD

1

C13

n-Tridecyl

A

1635

154

0.0132

5.81

2.97

2

C14

Isotetradecyl

D

1699

168

0.0110

1.37

1.30

3

C14

Anteisotetradecyl

D

1706

168

0.0015

0.88

1.14

4

C14

n-Tetradecyl

A

1737

168

0.0105

8.66

6.01

5

C15:1

Pentadecenyl 1, (8-)e

B,C

1816

208

0.0085

2.15

1.54

6

C15:1

Pentadecenyl 2, (10-)

C

1824

208

0.0075

1.38

0.93

7

C15

n-Pentadecyl

A

1838

182

0.0095

40.59

13.22

8

C16

Isohexadecyl

D

1900

196

0.0055

0.42

0.44

9

C16

Anteisohexadecyl

D

1909

196

0.0011

0.67

1.15

10

C16:1

Hexadecenyl 1, (7-)

B

1914

222

0.0082

0.46

0.32

11

C16:1

Hexadecenyl 2

D

1921

222

0.0096

0.06

0.10

12

C16

n-Hexadecyl

A

1939

196

0.0040

3.56

2.01

13

C17:2

Heptadecadienyl

B

2002

280

0.0071

2.94

1.59

14

C17:1

Heptadecenyl 1, (7-)

B,C

2014

236

0.0095

8.99

4.31

15

C17:1

Heptadecenyl 2, (8-)

C

2019

236

0.0089

2.06

1.17

16

C17:1

Heptadecenyl 3, (10-)

C

2029

236

0.0074

3.26

1.92

17

C17

n-Heptadecyl

A

2040

210

0.0027

5.33

3.26

18

C18:1

Octadecenyl 1

D

2085

296

0.0019

0.17

0.28

19

C18:2

Octadecadienyl

D

2100

294

0.0061

0.29

0.37

20

C18:1

Octadecenyl 2

B

2113

250

0.0041

0.92

0.93

21

C18:1

Octadecenyl 3, (9-)

A

2117

250

0.0041

(20+21)f

22

C18:1

Octadecenyl 4

D

2125

250

0.0056

0.31

0.40

23

C18

n-Octadecyl

A

2141

224

0.0017

0.49

0.58

24

C19:2

Nonadecadienyl 1

D

2198

308

0.0054

0.82

0.88

25

C19:2

Nonadecadienyl 2

D

2205

308

0.0065

0.46

0.60

26

C19:1

Nonadecenyl 1g

C

2215

264

0.0073

4.24

5.96

27

C19:1

Nonadecenyl 2, (12-)

C

2223

264

0.0067

1.63

1.93

28

C19:1

Nonadecenyl 3, (14-)

C

2233

264

0.0059

1.43

1.48

29

C19

n-Nonadecyl

D

2243

238

0.0019

0.60

0.78

aLevels of evidence: A, matched reference standard; B, matched literature KI calculated from the data of Garcia-Rubio et al. (2002); C, double bond position from DMDS data; D, MS and KI interpretation

bQI and QI/TIC are defined in Table 1 footnotes

cRelative amount is the proportion of all 29 formate esters, calculated by the ratio of the TIC to the sum of all TICs

dMean and SD values from four females and seven males. The remaining animals had no formate esters

eDouble bond position

fCompounds 20 and 21 were incompletely resolved and quantitated together

gDMDS adducts showed two isomers, Δ9- and 10-

Alcohol Moieties

Figure 4 shows the chromatograms for the TIC and m/z 47, as well as ion chromatograms that are relatively selective for the saturated and unsaturated formate esters. The chromatograms show the progressive elution of formate esters of longer carbon chain alcohols, with the unsaturated isomers eluting before the normal alkanyl compounds. The plotted ions were not uniquely identifying, since for example m/z 222 was formed from the C16:1 formates as [M-46]+. and C18:1 as [M-74]+.. Similarly, m/z 224 was [M-74]+. from the C18:0 formate, and [M-46]+. from the C16:0 formate. This lack of specificity was not confusing, as identification was based on relative retention time as well as the full mass spectrum. In some cases, peaks included more than one isomer, as for compound 20, which was not fully resolved from compound 21, and the two were quantitated together.

The formate esters of n-C13-18 and C18:1(9) (oleyl) alcohols were synthesized, and their MS and KI data confirmed the identification of these compounds. Linear regression analysis of KI vs. carbon number for the seven n-formate esters in Table 4 gave r2 = 1.000, slope 101.2 ± 0.1, P < 0.001, df 1, 5. Although n-C19:0 formate was not synthesized, its experimental KI fits this regression perfectly.

The branching of C14 esters 2 and 3, and C16 esters 8 and 9, appears to correspond to a methyl substituent at the ω-2 (iso) and ω-3 (anteiso) positions, respectively. Evidence for this branching was shown by the differences in losses of propyl (43) and ethyl (29) from the ions [M-46]+. and [M-74]+. (Supplementary Table 7). The first-eluting compounds of each pair of isomers (2 and 8) showed the ions that are expected for the iso isomer, corresponding to a loss of 43 rather than 29. The second-eluting isomers (3 and 9) showed a greater loss of 29, as expected for the anteiso isomer.

Double Bond Position

All of the n-alcohols present in formate esters also were found as the free alcohols in the cloacal samples, but neither the branched nor the unsaturated alcohols were detected, even after hydrolysis. This may be due to the large number of compounds present and the similarity of MS for fatty acids and alcohols. However, DMDS adducts provided data on the position of double bonds in some of the unsaturated formate esters (Supplementary Table 8). The EI mass spectrum of the DMDS adduct shows a pair of ions formed by cleavage of the bond between the thiomethyl groups, one corresponding to the formyl portion and the other to the alkyl portion of the molecule; and the sum of their m/z values equals M+ (Carlson et al., 1989; Mo et al., 1995). The M+ ion of the adduct was not always strong, but coelution with two thiomethyl fragment ions was good evidence for the adduct. The formate portion gave a confirmatory ion through the loss of 46 (formic acid).

A search for the DMDS adducts found these derivatives of some, but not all, of the alkenyl formates, but none for the dienes. The DMDS adducts eluted in the region of the chromatogram, which was dominated by decyl esters, and this interference may explain the failure to detect all of the expected adducts. Conversely, the two isomers that were unresolved in peak 26 (shown by a slight splitting of the m/z 47 peak in Fig. 4) were sufficiently resolved as DMDS adducts for their different double bond positions to be discerned. Table 4 gives the double bond positions for several of the alkenyl formates.

Since esters of two dimethyloctanols with many fatty acids were a major part of the cloacal samples, a search was made for evidence of formate esters with these alcohols. The search used ions characteristic of both decyl esters (m/z 140, 141) and formate esters (m/z 47, [M-46]+., [M-74]+.) and focused on the expected retention times (KI 1200-1300). A useful synthetic analogue was 3,7-dimethyloctyl formate (KI 1245). However, no evidence of a decyl formate was found.

Quantitation of Formate Esters

The median number of formate esters found per animal was 20 (range 0–28), with no sex difference. Four females and seven males had formate esters present, and these were used to calculate mean data (Table 4). n-Pentadecyl formate (7) was by far the major component, representing 40 % total formate esters, although this varied from 20 % to 65 % among individuals. The relative amounts of all of the formate esters showed great variability among individual possum samples, as shown by the large SD values. Other prominent components were heptadecenyl formate 1 (14), n-tetradecyl formate (4), n-tridecyl formate (1), and n-heptadecyl formate (17). Nearly half of the compounds were present at relatively low levels (<1 %).

Group 4: Sulfur Compounds

Initial Identification

Sulfur compounds were readily found, as they usually gave a strong molecular ion with the characteristic 34S isotope two units heavier with an isotopic abundance of about 4 % relative to the major 32S ion for each sulfur atom in the molecule (Beynon et al., 1968). Simple sulfur compounds (1–5 and 8 in Table 5 and Supplementary Table 9) were readily identified by comparison with published MS and KI data (NIST, 2008) whereas many of the polysulfides required first principles interpretation, and could not be uniquely identified in the absence of reference standards. Elemental sulfur (S8, compound 39) also was detected. The most volatile compounds, especially carbon disulfide, were readily lost from samples and were not always seen on re-analysis.
Table 5

Sulfur compounds found in possum cloacal swabs

Compound

LEa

KI

QIb

QI/TICb

Relative amount (%)c

No.

Name

Meand

SD

1

Carbon disulfide

B

632

76

0.467

0.60

1.01

2

Propanethiol

B

650

76

0.271

3.23

1.97

3

2-Methyl-1-propanethiol

B

684

90

0.157

0.76

0.52

4

2-Methylpropyl methyl sulfide

B

768

104

0.153

0.02

0.05

5

2-Methyl-1-butanethiol

B

792

104

0.103

0.24

0.36

6

n-Methyl propyl disulfide

B

931

122

0.232

0.77

0.61

7

Methyl isobutyl disulfidee

B

984

136

0.117

0.20

0.31

8

Dimethylsulfone

B

995

94

0.307

0.25

0.47

9

n-Ethyl propyl disulfide

B

1015

136

0.280

1.59

2.30

10

Ethyl isobutyl disulfidee

C

1069

150

0.146

0.31

0.60

11

n-Dipropyl disulfide

A

1110

150

0.238

10.57

7.73

12

n-Methyl propyl trisulfide

B

1154

154

0.297

1.36

0.79

13

Propyl isobutyl disulfidee

C

1161

164

0.159

3.49

2.32

14

Methyl butyl trisulfidee

C

1207

168

0.218

0.17

0.16

15

n-Propyl butyl disulfide

B

1209

164

0.230

0.02

0.03

16

Diisobutyl disulfidee

C

1210

178

0.206

0.11

0.13

17

n-Ethyl propyl trisulfide

B,C

1237

168

0.218

3.62

2.00

18

Propyl isopentyl disulfidee

C

1266

178

0.051

3.44

2.29

19

Ethyl butyl trisulfidee

C

1289

182

0.181

0.70

0.42

20

Isobutyl pentyl disulfidee

C

1314

192

0.048

0.22

0.33

21

n-Dipropyl trisulfide

B

1332

182

0.232

28.89

15.51

22

Propyl butyl trisulfidee

C

1382

196

0.173

11.10

4.38

23

Ethyl pentyl trisulfidee

C

1396

196

0.173

0.16

0.18

24

n-Methyl propyl tetrasulfide

C

1401

186

0.130

0.12

0.17

25

Ethyl butyl tetrasulfidee

C

1401

214

0.130

0.03

0.06

26

n-Propyl butyl trisulfide

C

1431

196

0.173

0.20

0.16

27

Dibutyl trisulfidee

C

1431

210

0.090

1.78

1.27

28

n-Propyl heptyl disulphide

B

1477

206

0.045

0.02

0.04

29

n-Ethyl propyl tetrasulfide

B

1484

200

0.130

0.16

0.20

30

Ethyl butyl tetrasulfidee

C

1484

214

0.130

0.05

0.08

31

Propyl pentyl trisulfidee

C

1489

210

0.090

6.07

3.05

32

Butyl pentyl trisulfidee

C

1537

224

0.093

1.13

0.86

33

n-Dipropyl tetrasulfide

B

1576

214

0.130

7.82

6.29

34

Propyl butyl tetrasulfidee

C

1622

228

0.130

0.18

0.19

35

Dibutyl tetrasulfidee

C

1622

242

0.042

0.23

0.25

36

Dipentyl trisulfidee

C

1643

238

0.037

0.36

0.53

37

Dibutyl tetrasulfidee

C

1667

242

0.042

0.14

0.27

38

n-Propyl heptyl trisulfide

C

1704

238

0.026

0.50

1.19

39

Sulfur

B

2078

256

0.092

9.36

19.39

aLevels of evidence: A, matched reference standard; B, matched literature MS and/or KI; C, MS and KI interpretation

bQI and QI/TIC are defined in Table 1 footnotes

cRelative amount is the proportion of all 39 sulfur compounds, calculated by the ratio of the TIC to the sum of all TICs

dMean and SD values from eight males and three females. Two males which had no sulfur compounds, and two females which had few, were excluded

eBranched/other isomers are possible

Dialkyl Polysulfides

Exploration of the TIC found several dialkyl di-, tri-, and tetrasulfides, and many other members of each series were readily located using chromatograms of the molecular ions. Figure 5 shows this for trisulfides from C4 to C10. The sulfide structure and sulfur number were supported by the 34S isotope ion and the presence of Sx and SxH2 ions. The alkyl substituents were identified by their R+ ions, except for the low mass ions (< m/z 35) that were not scanned. However, methyl and ethyl substituents produced related ions ([CH3S]+, [CH3S2]+, [C2H5S]+, and [C2H5S2]+), which confirmed their presence. Also, indirect evidence of the alkyl substituents was obtained since the polysulfides underwent a rearrangement to lose a neutral alkene molecule to form the ion [RSxH]+. (seen with propyl and above). The larger alkyl group was preferentially lost, and the major ion formed contained the smaller alkyl group. This provided additional evidence for the alkyl substituents in each compound, although not their isomeric forms. Where there was more than one isomer, the earlier eluting compounds were considered to be branched on one or both alkyl chains. For example, compound 7 is branched, since it eluted before its n-isomer (9) and, in addition to ions related to the methyl and butyl groups, showed a loss of C3H7 (to give m/z 93) but no loss of C2H5, indicating that the butyl group as branched. Methyl isobutyl disulfide gave the best match with NIST, but other branched isomers are possible.
https://static-content.springer.com/image/art%3A10.1007%2Fs10886-012-0188-5/MediaObjects/10886_2012_188_Fig5_HTML.gif
Fig. 5

Chromatogram showing peaks for the dialkyl trisulfides in Table 5. The cloacal sample was from a male possum. The top panel shows the total ion current (TIC), and the other panels show chromatograms of the molecular ions for dialkyl trisulfides with total carbon number from C4 to C10

Disulfides

Dialkyl disulfide compounds from C4 to C10 (Supplementary Table 10) gave significant molecular ions, which became less abundant at higher molecular weights, as previously reported (Gupta et al., 1981). The disulfide structure was confirmed by the 34S isotope ion, which was about 9 % of the 32S ion, and ions for [S2]+. and [S2H2]+. (m/z 64 and 66, respectively). There were sufficient data on alkyl ions and alkene losses to assign the carbon numbers of the alkyl groups, even if their structural isomers could not be completely elucidated. Compound 28 had the expected KI for the unbranched isomer (Fig. 6), and showed ions corresponding to a heptyl group ([C7H5S]+m/z 131; [C7H5S2]+m/z 163). The low abundance of the heptyl ion has previously been noted in n-diheptyldisulfide (Gupta et al., 1981).
https://static-content.springer.com/image/art%3A10.1007%2Fs10886-012-0188-5/MediaObjects/10886_2012_188_Fig6_HTML.gif
Fig. 6

Plots of Kovats Index (KI) vs. carbon number (Cn) for dialkyl disulfides (S2), trisulfides (S3) and tetrasulfides (S4). The regression lines are for the n-isomers and all showed excellent linearity (r2 > 0.999). Compounds falling below the lines are considered to be branched isomers. The slopes of the regression lines did not differ significantly (P = 0.394) although the intercepts differed (S2 = 561 ± 7; S3 780 ± 7; S4 1050 ± 13; P < 0.001, df 2,9). The overall slope was 92.1 ± 0.7 KI/carbon number (P < 0.001, df 1, 11)

Trisulfides

Dialkyl trisulfides from C4 to C10 were initially detected from the mass chromatograms of their molecular ions (Fig. 5). There was a strong 34S isotope, mostly about 12 % of the 32S ion, consistent with the presence of three sulfur atoms (Supplementary Table 11). The most abundant sulfur ions were [S3H2]+. and [S2]+., with smaller amounts of [S3]+. and [S2H2]+.. As for the disulfides, the alkyl groups were identified by their R+ ions, and indirectly by the loss of the corresponding olefin to give [R1S3H]+. and, to a lesser extent, [R2S3H]+. (where R1 is the smaller alkyl group). Some compounds also formed ions corresponding to [RS]+, [RSH]+., [RS2]+, and [RS2H]+..

Compounds 12 and 21 had KI values similar to the literature values for the unbranched isomers. The KI of 17 (ethyl propyl trisulfide, 1237) was similar to that of another unbranched C10 trisulfide (n-methyl butyl trisulfide, KI 1241), suggesting that it is also unbranched. These unbranched isomers 12, 17, and 21 lay on a linear regression line of KI vs. Cn, which included compounds 26 and 38, and paralleled similar regression lines for disulfides and tetrasulfides (Fig. 6), indicating that all these compounds are unbranched on both alkyl substituents.

In dipentyl trisulfide (36), the absence of the expected [C5H11S3H]+. (m/z 168) ion was possibly due to its subsequent fragmentation to [C5H11S2]+ (m/z 135), [C5H11SH]+. (m/z 104), and [C5H11S]+ (m/z 103). Propyl butyl trisulfide and dibutyl trisulfide (26 and 27) co-eluted and gave a combined mass spectrum. Since a loss of 14 mass units is not possible in a mass spectrum, the ions at m/z 210 and 196 were considered to be the molecular ions of two compounds, and their fragmentations were interpreted accordingly. Some ions (m/z 57, 64, 98, 154) were common to both compounds, and their contributions could not be separately apportioned. The presence of the dibutyl compound probably caused distortion in relative abundances of [C3H7S3H]+. and [C4H9S3H]+. for propyl butyl trisulfide, as well as suppression of its molecular ion (m/z 196).

Tetrasulfides

The mass spectra of dialkyl tetrasulfides provided abundant data to assist their identification (Supplementary Table 12). The molecular ions showed compounds with substitution from C4 to C8, and the +2 isotope ion, mainly from 34S, was prominent at 15–27 % of the 32S ion. Variations between the calculated and actual values for the isotopic abundances are explained by the relatively low signal to noise in some spectra. Other ions diagnostic for alkyl tetrasulfides were [S4]+. and sometimes [S4H2]+., and [R1S4H]+. and [R2S4H]+., although in this case (unlike the di- and trisulfides) the smaller alkyl fragment was not always predominant. Further evidence for the alkyl substituents was given by the rearrangement ions [R1S2H]+. and [R2S2H]+., which were generally more abundant than the corresponding [RS4H]+. ions, and various alkyl sulfur ions ([RS]+, [RS2]+, [RS2H]+., and [RS3]+.).

Compounds 24 and 25 co-eluted and consequently gave a combined mass spectrum; however, there was abundant evidence for four alkyl groups, which indicated that there were two compounds with molecular weights 186 (C4H10S4) and 214 (C6H14S4). The four fragment ions for both RS4H and RS2H showed that there were methyl, ethyl, propyl, and butyl groups present, and there were confirmatory R+ ions for the propyl and butyl groups. The molecular weights gave the two compounds as methyl propyl and ethyl butyl tetrasulfide. The alkyl groups were confirmed by other prominent ions that were assigned to compound 24 ([CH3S1-3]+, [CH3S3H]+., [C3H7S1-3]+, [C3H7SH]+.) and to compound 25 ([C2H5S]+, [C4H9S]+, [C4H9SH]+., [C4H9S3H]+.).

Two other pairs of compounds also co-eluted, 29 and 30, and 34 and 35. Like the trisulfides 26 and 27, each pair showed molecular ions separated by 14 mass units, indicating that two compounds were present. Their alkyl substituents were found from the S-adduct ions, enabling the structural assignments. The regression plot of KI vs. carbon number (Fig. 7) indicated that compounds 24, 29, and 33 were the only unbranched tetrasulfides.

For each set of polysulfides, the n-isomers showed an excellent linear regression, and the lines were parallel, thus supporting the identifications (Fig. 6). The branched isomers eluted earlier and, since their branching was unknown and probably variable, regression analysis was not appropriate. There was a remarkable regularity in the structures of the unbranched homologous dialkyl polysulfides (Table 5). The C4 compounds were all methyl propyl isomers, C5 were ethyl propyl, C6 were dipropyl, C7 were propyl butyl, C8 and C9 were missing, and C10 were propyl heptyl.

Quantitation of Sulfur Compounds

The median number of compounds found per animal was 18 (range 0–39), and there was no sex difference. Two males (one a juvenile) had no sulfur compounds present, and two females had few (6 and 7) compounds. The remaining 11 animals had 17–39 compounds present, and these were used to calculate mean data (Table 5). Among the major components were dipropyl disulfide (11 %), dipropyl trisulfide (30 %), dipropyl tetrasulfide (8 %), and elemental sulfur (9 %). There were 14 compounds with mean relative amounts greater than 1 %, and these accounted for 95 % of the total of all compounds. Having few compounds means that their relative percentages are high, and the two excluded female possums had large proportions of dimethylsulfone (50 % and 96 %), far more than found in the possums with a fuller range of compounds (0.3 %).

Group 5: Alkylglycerol Ethers

A series of 1-O-alkylglycerol ethers were found as non-saponifiable compounds after hydrolysis of the acylglycerides. They produced significant TIC peaks as their di-TMS derivatives, with characteristic ions at m/z 205 (loss of CH2OR), 103 ([CH2OTMS]+), 147 (a bis-TMS ion), 133, 117, and 73, as reported previously (Khannoon et al., 2011). The molecular ion usually was absent, but there were ions at [M-15]+ and a stronger ion at [M-147]+ that gave the molecular weight. The series included n-alkyl compounds from C13 to C20 with some methyl-branched isomers. The major compounds were C16, C15, and C17. For the n-acyl compounds, linear regression of KI vs. Cn gave a slope of 95.4 ± 0.1, r2 = 1.000, P < 0.001 (N = 8). The KI of 1-O-hexadecylglycerol, bis-TMS ether was 2474. These alkylglycerol ethers were present mainly in the cloaca in the esterified form as the glycerides of fatty acids, and they were not further investigated or quantitated.

Discussion

This study sought to identify the compounds which are deposited when a possum marks an object with its cloaca, by analyzing swabs of the cloacal surface. Although it was beyond the scope of the study to explore the source of compounds found, it is assumed that the major portion originated in the cloacal glands, but contributions could come from urine, feces, or possibly the apocrine and sebaceous glands that are present in the groin (Green, 1963). The chemical complexity could have been increased by bacterial fermentation as occurs in the anal sacs of some mammals (Alberts, 1992). Compounds that are normal biochemicals (e.g., lactic acid, cholesterol) or which have been found previously in possums as urinary metabolites of dietary terpenes and phenolics (Boyle et al., 2000; McLean et al., 2003) were not considered to be primarily scent chemicals and were not examined in detail. No possum produced all of the cloacal chemicals found, but neither did any animal produce none. All produced acids and alcohols, and two males (one a juvenile) produced only these compounds. Three animals only missed producing decyl esters, one missed the formates, and another missed both. However, in some cases where a compound was missing, there had been a sub-threshold peak, and re-analysis of a larger sample may have given a positive finding. As in any analytical study, missing compounds may in some cases have been simply below the detectable limit.

The 81 free fatty acids and alcohols also were partly present in esterified form, and could be the products of hydrolysis by skin bacteria (Shalita, 1974). However, regardless of their source, these classes of compounds are known to be able to act as pheromones (Wyatt, 2003), and therefore have the potential to make an important contribution to possum chemical communication. The C7-C9 branched acids previously have been found in possum cloacal holocrine glands, while longer chain unsaturated acids have been identified as acyl glycerides in the cloacal apocrine glands (Woolhouse et al., 1994). However, the extended homologous series of normal, branched and unsaturated acids and alcohols found in the present study appears to be a unique suite of scent chemicals.

Burger and colleagues have made extensive investigations of the chemistry of exocrine secretions of several species of antelope and other eutherian mammals, and have found a large number of fatty acids and alcohols and their esters. For example, preorbital secretions of the steenbok (Raphicerus campestris) contained mostly long-chain, unbranched, saturated and unsaturated alcohols as well as some saturated and unsaturated fatty acids (Burger et al., 1999a). The ventral gland secretions of the male dwarf hamster (Phodopus sungorus sungorus) yielded saturated and unsaturated straight chain acids, and iso- and anteiso acids as well as saturated alcohols (Burger et al., 2001). A number of fatty acids and alcohols were included in the marking fluid of the male Bengal tiger, Panthera tigris (Burger et al., 2008), and a large number of unsaturated alcohols were found in the preorbital secretion of the grysbok, Raphicerus melanotis (Burger et al., 1996). The preorbital glands of the sika deer (Cervus Nippon) contain straight- and branched-chain fatty acids (Wood, 2003). Several C14–C16 unsaturated and branched alcohols, free and esterified, have been found in mouse preputial glands and have been associated with both individual and strain differences (Zhang et al., 2007). Some C12–C18 fatty acids have been found in subcaudal gland secretions of the European badger, Meles meles (Buesching et al., 2002). A more comprehensive review of the literature can be found in Burger (2005).

The 2,6- and 2,7-dimethyloctanols appear to be new to mammalian chemistry, and this is the first report of their extended set of fatty acid esters. A set of esters of C14–C17 alcohols and C6–C12 acids has been reported in the collared peccary, Tayassu tajacu (Waterhouse et al., 1996). Hydroxyalkyl esters of acetate and butyrate are found in the steenbock (Burger et al., 1999a), and acids from acetic to pentanoic in the bontebok (Damaliscus dorcas dorcas) and blesbok (D. d. phillipis) (Burger et al., 1999b). However, there have been no reports of esters similar to the branched decyl esters of saturated and unsaturated acids from C7 to C20 that we found in the brushtail possum.

Formate esters are not novel semiochemicals, although this is their first report in a marsupial. Formate esters are scent chemicals in the periorbital secretions of several species of antelope, which are used for territorial marking (Burger, 2005). The grysbok secretes a large number of unbranched, long-chain saturated and unsaturated alcohols and their formate esters: C11–C25 alkyls, C13–C25 alkenyls and C14–C21 alkadienyls, including compounds 5, 15, and 25 reported from this possum study (Burger et al., 1996). The oribi (Ourebia ourebi) secretes a smaller number of formates: C8–C16 alkyls and C9–C15 alkenyls, and one branched compound, 13-methyl-(Z)-8-pentadecyl formate, an anteiso isomer (Mo et al., 1995). The steenbok (R. campestris) secretes unbranched C11–C28 alkyl and C11–C15 alkenyl formates (Burger et al., 1999a) and the suni (Neotragus moschatus) secretes unbranched C11–C24 alkyl and C13–C25 alkenyl formates (Stander et al., 2002). In addition, one reptile, the American crocodile (Crocodylus acutus) has C12–C20 straight chain alcohols and their formate, acetate and butyrate esters in its paracloacal glands (Garcia-Rubio et al., 2002).

Organosulfur compounds have been identified as scent chemicals in several mammalian species, but no other animal has shown the extended sets of homologous polysulfides that are present in the brushtail possum. Sulfur compounds are prominent in the anal sacs of two related families of carnivorous mammals, the Mephitidae (e.g., skunks) and Mustelidae (e.g., weasels), which are well-known for their offensive smell. In four species of North American skunks (Mephitis spp.), the major sulfur compounds consist of thiols (2-butene-1-thiol; 3-methyl-1-butanethiol; 2-phenylethanethiol; 2-quinolinemethanethiol) and thioacetates (2-butenyl thioacetate; 3-methylbutanyl thioacetate), and there were traces of some disulfides (e.g., 2-butene-3-methylbutyl disulfide) (Wood et al., 2002). Substituted thietanes and dithiolanes are found in the stoat (Mustela ermine) and ferret (M. putorius), as well as one open chain compound, diisopropyl disulfide, in the ferret (Crump, 1980a, b; Crump and Moors, 1985). Thietanes and dithiolanes also have been identified in other mustelids (Brinck et al., 1983; Zhang et al., 2002). In addition to these cyclic sulfides, the mink (M. vison) has several open chain disulfides: dimethyl disulfide; dibutyl disulfide; isopentyl methyl disulfide; butyl 3-methylbutyl disulfide; and bis(3-methylbutyl) disulfide (Sokolov et al., 1980). Most of the anal gland chemicals of the ferret (including the thietanes and dithiolanes) were not found among urinary volatiles, although benzothiazole is found in both (Zhang et al., 2005).

Two small antelope species produce sulfur compounds in their scent glands. The grysbok secretes a black aqueous emulsion of organic compounds from the preorbital gland containing, among 90 other compounds (alcohols, fatty acids, formates, aldehydes, and lactones), two dialkyl sulfides: methyl heptadec-1-yl sulfide and methyl nonadec-1-yl sulfide (Burger et al., 1996). The preorbital secretion of the red duiker (Cephalopus natalensis) contains alcohols, aldehydes, fatty acids, ketones, spiroacetals, and two thiazoles: 2-isobutyl-1,3-thiazole and 2-isobutyl-4,5-dihydro-1,3-thiazole (Burger, 2005).

Sulfur compounds are found in the urine of several species. Sulfides and disulfides have been detected in the female beagle, Canis lupus familiaris (Schultz et al., 1985) and coyote, Canis latrans (Schultz et al., 1988), which also contained dimethyl trisulfide. The strong odor of the red fox (Vulpes vulpes) has been linked to delta-3-isopentenyl methyl sulfide and 2-phenylethyl methyl sulfide (Jorgenson et al., 1978). Among the great number of urinary volatiles found in the mouse are dimethylsulfide and 2-sec-butyl-4,5-dihydrothiazole (Schwende et al., 1986). In a more recent update, a number of other sulfur compounds were identified, including bis(methylthio)methane and methyl (methylthio)methyl disulfide (Novotny et al., 2007). Elemental sulfur is found in the urine of the cheetah (Acinonyx jubatus) (Burger et al., 2006) and in marking fluid of the male Bengal tiger (Burger et al., 2008) together with traces of dimethylsulfone and, in the cheetah, dimethyl disulfide. A novel α-diketone thioether, 5-thiomethylpentane-2,3-dione, has been reported in the anal gland of the striped hyena (Hyaena hyaena) (Wheeler et al., 1975). Many plants also contain polysulfides and other sulfur compounds, and some have found uses as spices in human culinary dishes, and as folk medicines (Kuo and Ho, 1992; Pino et al., 2001; Lee et al., 2003).

Many of the sulfur compounds found in the cloaca of the brushtail possum appear to be new to biology, and certainly are previously unidentified in mammals. The novel biological compounds are disulfides 18 and 28; trisulfides 22, 23, 26, 31, 32, and 38; and tetrasulfides 25, 29, 30, 34, 35, and 27.

Alkylglycerol ethers have been found in terrestrial animals as diacylglycerol ether lipids and phosphoether lipids, although they are most abundant in Elasmobranch fish (Magnusson and Haraldsson, 2011). In mammals, they are present as minor constituents of cell membranes and also as mediators (e.g., platelet activating factor) of many biological effects on cell growth, immunity, reproduction, and circulation (Iannitti and Palmieri, 2010; Magnusson and Haraldsson, 2011). This supports the view that they may have semiochemical activity. Free alkyl diacylglycerols (especially 1-O-hexadecylglycerol) have been found in the secretion of the western diamond rattlesnake, Crotalus atrox (Weldon et al., 2008). Several of the possum samples had significant peaks for free alkylglycerol ethers, especially the C16 compound, but the major amount was released after hydrolysis. In non-primate mammals, the Harderian gland, which is located in the orbit, secretes a lubricating fluid that contains alkyl diacylglycerols, which are likely to have a semiochemical role in some species (Kasama et al., 1970; Yamazaki et al., 1981; Albone, 1984; Harvey, 1991). Further investigation of the alkyl glycerol ethers in the possum will require identification of the parent esterified forms. This was beyond the scope of the present investigation.

We considered investigating sex differences in chemical profiles by using multivariate statistical methods (discriminate or principal component analyses) but the data, which were collected as a first description of possum scent chemicals, were inadequate for this purpose. This was primarily because the number of possum samples was small relative to the large number of chemical variables found, and the presence of many values that were undetected or zero can seriously bias a regression analysis (Helsel, 2005). Additionally, regression analysis assumes that the variables are independent, but many of the chemicals are likely to be related (homologues; alcohols and their corresponding acids; esters and their acid and alcohol moieties), and this multicollinearity weakens the assumption of independence (Martin and Drijfhout, 2009). Future studies will need to use larger numbers of samples, improve detection limits, and examine the correlations between variables in order to enable multivariate regression analysis of the relationship between chemical and biological variables.

Although there have been few studies of chemical communication in marsupials, the available evidence indicates that in this they are similar to eutherians. For example, the vomeronasal organ of the tammar wallaby (Macropus eugenii) shows a typical mammalian structure, suggesting that this organ developed in a common ancestor of all mammals (Schneider et al., 2008). The male antechinus (Antechinus agilis), a small carnivorous marsupial, uses paracloacal and other secretions to scent mark during the breeding season, and females use these scents to choose genetically-dissimilar mates (Parrott et al., 2007). The koala (Phascolarctos cinereus) scent marks with the sternum, whose secretions include fatty acids, alcohols, aldehydes, and ketones (Salamon and Davies, 1998; Tobey et al., 2009).

A complex mixture of scent chemicals is able to carry more information than a smaller number of compounds. For example, a range of volatilities enables both a rapid onset of signal and slow fade-out; and the more volatile components can produce an airborne concentration gradient, signaling from a distance and indicating the direction of the marked location, as well as the age of the mark (Alberts, 1992). Variations in relative amounts of constituents can produce individual scent profiles.

The cloacal chemicals found here differed among possums in their relative amounts, as well as their presence or absence, and may be acting to identify a particular individual or family group rather than as classical pheromones. Wyatt (2010) has proposed the term “signature mixture” for this form of chemical communication, to distinguish it from classical pheromones in which a standard chemical (or particular combination of chemicals) elicits a specific, innate response in a receiving individual of the same species. A signature mixture is a variable subset of an animal’s total chemical profile, which other animals learn to associate with a particular individual or group (family or colony). Pheromonal signals must be recognized against this background, as all chemicals are detected and processed by the same olfactory systems, the main olfactory system and the vomeronasal system (Tirindelli et al., 2009). Individual recognition is essential to avoid conflicts with animals of different status or strangers, and to prevent mating with close kin (Biggins, 1984; Wyatt, 2010). Elucidation of the role of individual chemicals in communication will require studies of their association with mating and other behaviors. Possums exhibit complex social behavior, which probably depends in large part on chemical communication (Biggins, 1984; Spurr and Jolly, 1999; Day et al., 2000). Further investigation into the biological production and use of cloacal compounds in the brushtail possum will assist us in unraveling their potential role in intraspecific (and interspecific) chemical communication.

Acknowledgements

We thank Stephen Quarrell for assistance with the DMDS derivatization reaction. We are grateful to Kathleen R. Murphy for advice on the feasibility of multivariate regression analysis with this dataset.

Supplementary material

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© Springer Science+Business Media, LLC 2012