Reptilian chemistry: volatile compounds from paracloacal glands of the American crocodile (Crocodylus acutus).

Article abstract of DOI:10.1023/A:1015288726605

The secretion of the paracloacal glands of the American crocodile (Crocodylus acutus) contains over 80 lipophilic compounds, including saturated and unsaturated long-chain alcohols along with their formic, acetic, and butyric acid esters, and several isoprenoids. Most of these compounds were identified on the basis of mass spectra, obtained by GC-MS. In addition, identification of the major components was supported by infrared spectra obtained by GC-FTIR. Major differences are indicated in the composition of the paracloacal gland secretion of C. acutus and that of another crocodylid, the African dwarf crocodile (Osteolaemus tetraspis).

Full text of DOI:10.1023/A:1015288726605

C
Journal of Chemical Ecology, Vol. 28, No. 4, April 2002 ( 2002)
REPTILIAN CHEMISTRY: VOLATILE COMPOUNDS FROM
PARACLOACAL GLANDS OF THE AMERICAN CROCODILE
(Crocodylus acutus)
1
1
´
SILVINA GARCIA-RUBIO, ATHULA B. ATTYGALLE,
PAUL J. WELDON,2 and JERROLD MEINWALD
,1
1Department of Chemistry and Chemical Biology
Cornell University
Ithaca, New York 14853-1301
2Conservation and Research Center
Smithsonian Institution
1500 Remount Road
Front Royal, Virginia 22630
(Received April 16, 2001; accepted December 5, 2001)
Abstract—The secretion of the paracloacal glands of the American crocodile
(Crocodylus acutus) contains over 80 lipophilic compounds, including saturated
and unsaturated long-chain alcohols along with their formic, acetic, and butyric
acid esters, and several isoprenoids. Most of these compounds were identified
on the basis of mass spectra, obtained by GC-MS. In addition, identification
of the major components was supported by infrared spectra obtained by GC-
FTIR. Major differences are indicated in the composition of the paracloacal
gland secretion of C. acutus and that of another crocodylid, the African dwarf
crocodile (Osteolaemus tetraspis).
Key Words—American crocodile, Crocodylus acutus, paracloacal glands, fatty
esters, formates, mass spectrometry, GC-FTIR.
INTRODUCTION
While insect chemical ecology has made great strides in the last five decades,
much less is known about many comparable aspects of vertebrate chemical ecol-
ogy (Eisner and Meinwald, 1995). The crocodilians, a small but fascinating group
of ancient vertebrates, have aroused the curiosity of chemical ecologists in the last
decade, with primary attention being paid to the chemical composition of their skin
To whom correspondence should be addressed. E-mail: circe@cornell.edu
769
C
0098-0331/02/0400-0769/0 2002 Plenum Publishing Corporation

´
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
770
gland secretions (Weldon and Wheeler, 2000). The study of crocodilian ecology
and behavior, however, has lagged behind that of most other tetrapods. This cir-
cumstance can be attributed in part to the difficulties inherent in studying responses
to chemicals by these large and, for adults at least, dangerous reptiles in field or
seminatural settings. We have studied the chemistry of the secretion produced in
the paracloacal glands of the American crocodile (Crocodylus acutus, Crocodyli-
dae) in the hope that knowledge of this chemistry will ultimately contribute to an
understanding of crocodilian chemical ecology. These glands in crocodilians are
thought to produce pheromones involved in the nesting and/or mating activities
(Weldon and Ferguson, 1993).
Previous analyses of lipids from the paracloacal glands of crocodilians have
occasionallyledtothecharacterizationofunusualcompounds, thusreflectingsome
novel biosynthetic capabilities of these reptiles (Weldon and Wheeler, 2000). For
example, a unique 19-carbon aromatic ketone, named dianeackerone, and a family
of related steroidal esters were found to be major glandular products of the African
dwarf crocodile (Osteolaemus tetraspis, Crocodylidae) (Whyte et al., 1999; Yang
et al., 1999, 2000). Two stereoisomers of dianeackerone occur in varying ratios in
adults of this species, but this compound was not detected in immatures.
We report here the results of an analysis by gas chromatography–mass spec-
trometry (GC-MS) of the paracloacal gland secretion of another crocodylid,
C. acutus. This species occurs in freshwater and brackish coastal habitats in
Mexico, Central America, northern South America, Cuba, Jamaica, Hispaniola,
and in the tip of southern Florida. Our study revealed that the glandular secretion of
this species is composed largely of formate, acetate, and butyrate esters of C12–C20
straight-chain alcohols, along with the alcohols themselves. Interestingly, formate
esters have not previously been reported in reptiles. This is the first detailed report
of the skin gland lipids of Crocodylus, the most speciose genus of crocodilians.
METHODS AND MATERIALS
Paracloacal gland secretions of C. acutus were obtained from one adult
male [total length (TL) = 3.0 m], one adult female (TL = 2.5 m), six juvenile
males (TL = 1–1.5 m), five juvenile females (TL = 0.7–1.1 m), and five un-
sexed hatchlings (TL = 0.5–0.6 m) maintained at Cypress Gardens (Plant City,
Florida), Gatorama (Palmdale, Florida), and the St. Augustine Alligator Farm
(St. Augustine, Florida). The adults we sampled, which had been housed together
for 2.8 years, are within the size range of reproductively active, free-ranging
C. acutus (Kushlan and Mazzotti, 1989); they had copulated, although neither
egg-laying nor nesting behavior had been observed. Most secretion donors hatched
from eggs laid by captive females from Florida; one juvenile female was reared
from an egg laid by a female from Jamaica. Animals at Cypress Gardens were
fed rats and chicken necks, those at Gatorama were fed a commercial feline food

CROCODILE VOLATILE COMPOUNDS
771
(Nebraska Brand, Central Nebraska Packing Company, North Platte, Nebraska)
containing primarily horse meat, and those at the St. Augustine Alligator Farm
were fed nutria (Myocastor coypu).
The secretions were collected by manually pressing the region around the
cloacal opening, causing the walls of the cloaca to flare outward, exposing the
openings of the gland ducts. Additional pressure applied to the glandular region
resulted in the discharge of yellow secretions from the glands. These exudates were
collected in capillary tubes that were broken off into glass vials. A few milliliters
of CH2Cl2 were added to the vials, which were stored on dry ice.
Determination of Position of Double Bonds. Dimethyl disulfide (DMDS)
derivatization was carried out according to a reported procedure (Attygalle and
Morgan, 1986).
Reference Compounds. Fatty acids and alcohols, cholesterol, cholesta-3,5-
diene, citronellol, pentadecane, and squalene were purchased from Sigma-Aldrich.
Cholesteryl and alkyl formates were synthesized by the uncatalyzed reaction of
each alcohol with an excess of formic acid (Burger et al., 1996). Acetates were
synthesized by treating each alcohol with an excess of acetic anhydride and pyri-
dine (Zhdanov and Zhenodarova, 1975). The reaction of the alcohols with bu-
tyryl, caproyl, and capryl chloride in CH2Cl2 afforded the corresponding butyrates,
caproates, and caprylates, respectively (Ihara et al., 1990; Madden and Prestwich,
1994). 2,3-Dihydrofarnesol was prepared following a literature procedure (Kaplay
et al., 1980).
Analytical Procedures. Gas chromatography was performed on a Hewlett-
Packard (HP) 5890 instrument equipped with a splitless injector, and either a
flame-ionization detector, a HP 5965A IR detector, or a HP 5970 mass selective
detector. Thesampleswereinjectedinthesplitlessmode. Analyseswereperformed
using a 25-m 0.22-mm fused silica-column coated with SE-54. The oven was
programmed from 40 C (2 min) at 7 C/min to 280 C (30 min). To investigate
the possible presence of nonvolatile compounds, we undertook a direct analysis of
unfractionated secretion samples by NMR spectroscopy, as well as by electrospray
ionization–mass spectrometry. No additional compounds were found with these
methodologies.
RESULTS AND DISCUSSION
An exploratory survey of the paracloacal gland secretions of 21 crocodil-
ians by thin-layer chromatography showed the presence of bands consistent with
aliphatic esters in most taxa, including Crocodylus spp. (Weldon and Tanner,
1991). Analyses by GC-MS have revealed the structures of these esters in alligators
(Alligator spp.) and caimans (Caiman spp. and Paleosuchus spp.) (Shafagati et al.,
1989; Dunn et al., 1993; Ibrahim et al., 1998). The present study confirms the
presence of many aliphatic acetates in C. acutus, and demonstrates the occurrence

´
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
772
of formates (characterized by m/z 47 ions) and butyrates (characterized by m/z 89
ions) as well. Moreover, the ester derived from 1-hexadecyl alcohol is the main
component of the caproic acid esters; 1-hexadecyl caproate (m/z 117 and 224 ions)
is also abundant in the secretions of the Chinese alligator (A. sinensis) (Dunn et al.,
1993). Our analytical results are presented in Tables 1 and 2 and Figures 1 and 2.
We observed significant quantities of formate esters derived from C13–C18
alcohols; the major formates were those of C16, C17, and C18 monounsaturated
alcohols. The presence of formates is interesting since they have not been reported
previously from crocodilians or from any other reptiles. While formates are known
from ants (Lopez et al., 1993), beetles (Attygalle et al., 1992), and caterpillars
(Attygalle et al., 1993), to the best of our knowledge they have not been reported
among vertebrates except in the preorbital gland secretions of three species of
African antelopes (Burger et al., 1981, 1996, 1999; Mo et al., 1995). The alcoholic
moieties of these esters range, among the three antelope species, from 9 to 28
carbons. As Burger et al. (1981) noted, the mass spectra of formates are very
similar to those of the corresponding aliphatic alcohols, but are distinguished
from them by the presence of a low-intensity peak at m/z 47, corresponding to an
+
HCO2H2 (protonated formic acid) fragment.
We observed free alcohols in the C. acutus ranging from 10 to 18 carbons,
including the terpene citronellol, which was found only in the secretion of the
hatchlings. A similar array of alcohols has been reported in some alligatorids, e.g.,
FIG. 1. A reconstructed gas chromatogram obtained from the paracloacal gland secretion
of hatchling Crocodylus acutus. Peak numbers refer to components listed in Table 1.

CROCODILE VOLATILE COMPOUNDS
773
TABLE 1. COMPONENTS FOUND IN PARACLOACAL GLAND SECRETION OF Crocodylus
acutusa
Molecular
formula
Characteristic ions
observed (m/z)
No.
Compound
NMM
Rt (min)
1
2
3
4
5
6
7
8
9
Citronellyl formate
Citronellyl acetate
Pentadecane
Unidentified terpenoid
Citronellyl butyrate
Dodecyl formate
Tridecyl alcohol
Dodecyl acetate
Tridecyl formate
Unidentified terpenoid
Tetradecyl alcohol
Unidentified terpenoid
Tetradecenyl formate1
Tridecyl acetate
Unidentified terpenoid
Tetradecyl formate
Myristic acid
Unidentified terpenoid
Pentadecyl alcohol
Tetradecenyl acetate1
Unidentified
C10H19OCHO
C10H19O2C2H3 198 138, 123, 95, 81, 69, 43, 41
184 138, 123, 95, 81, 69, 41
15.33
16.92
20.11
20.17
20.28
20.30
21.15
21.71
22.12
22.19
22.87
23.30
23.41
23.41
23.81
23.81
24.21
24.21
24.52
24.62
24.88
C15H32
212 212, 71, 57, 43
181, 135, 109, 69
C10H19O2C4H7 226 138, 123, 95, 81, 69, 43, 41
C
C
12H25OCHO
13H27OH
228 168
200 182
228 168
228 182
195, 123, 69, 41
214 196
209, 149, 123, 81, 69, 43, 41
240 194
C12H25OCHO
C13H27OCHO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
C14H29OH
C14H27OCHO
C13H27O2C2H3 242 182
209, 123, 69, 41
242 196
228 185
C14H29OCHO
C13H27CO2H
195, 143, 171
228 210, 182
C14H27O2C2H3 256 194
252 252, 135, 121, 79, 67,41
C15H31OH
Dihydrofarnesyl acetate C15H27O2C2H3 266 223, 163109, 95, 81, 69, 43, 41 24.93
Tetradecyl acetate
C14H29O2C2H3 256 196
25.01
25.01
25.42
25.71
25.71
25.97
26.11
26.18
26.23
26.39
26.55
26.55
26.80
26.94
27.15
27.22
27.38
27.48
27.48
27.61
27.61
27.64
Pentadecenyl formateb
Pentadecyl formate
Hexadecenyl alcoholb
Pentadecanoic acid
C15H29OCHO
15H31OCHO
254 208
256 210
240 222
242 199
C
C16H31OH
C14H29CO2H
Pentadecadienyl acetateb C15H27O2C2H3 266 206
Hexadecyl alcohol
Pentadecenyl acetateb
Tridecyl butyrate
Unidentified
C
16H33OH
C15H29O2C2H3 268 208
13H27O2C4H7 270 182
266 266, 135, 121, 81, 67, 41
268 222
242 224
C
7-Hexadecenyl formateb C16H31OCHO
Pentadecyl acetate
Methyl palmitate
Hexadecyl formate
Heptadecadienyl alcoholb C17H31OH
C
C
15H31O2C2H3 270 210
15H31CO2CH3 270 227, 270
C16H33OCHO
270 224
252 234
274 256
254 236
256 238
Palmitic acid
C15H31CO2H
C17H33OH
Heptadecenyl alcoholb
Heptadecyl alcohol
C17H35OH
Hexadecadienyl acetateb C16H29O2C2H3 280 220
Unidentified terpenoid
Hexadecenyl acetatec
Tetradecenyl butyrateb
251, 191, 163, 81, 69
C16H31O2C2H3 282 222
C14H27O2C4H7 282 194

´
774
No.
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
TABLE 1. CONTINUED
Molecular
formula
Characteristic ions
Compound
NMM
observed (m/z)
Rt (min)
45
46
47
48
49
50
51
Tetradecyl butyrate
C14H29O2C4H7
284 196
282 234
280 280, 135, 121, 81, 67, 41 27.87
284 224
282 236
284 238
268 250
27.69
27.78
Heptadecadienyl formateb C17H31OCHO
Unidentified
Hexadecyl acetate
C16H33O2C2H3
C17H33OCHO
28.02
28.02
28.42
28.70
28.77
28.81
28.87
28.87
29.02
29.10
29.10
29.10
Heptadecenyl formateb
Heptadecyl formate
Octadecenyl alcohold
C17H35OCHO
C18H35OH
52
53
54
55
Pentadecenyl butyrateb
Unidentified terpenoid
C15H29O2C4H7
296 208
169, 109, 69, 41
294 234
296 236
Heptadecadienyl acetateb C17H31O2C2H3
Heptadecenyl acetatee
C17H33O2C2H3
56
57
58
59
60
Octadecyl alcohol
Pentadecyl butyrate
Unidentified
Heptadecyl acetate
Octadecenyl formate f
C18H37OH
270 252
298 210
294 294, 135, 121, 81, 67, 41 29.28
298 238
296 250
C15H31O2C4H7
C17H35O2C2H3
C18H35OCHO
29.40
29.40
29.48
29.69
29.79
30.07
30.07
30.09
30.24
30.36
30.42
30.42
30.72
61
62
63
64
65
Oleic acid
C
17H33CO2H
C18H37OCHO
17H35CO2H
282 264
298 252
284 266
310 222
308 248
Octadecyl formate
Stearic acid
Hexadecenyl butyrateb
Octadecadienyl acetateb
C
C16H31O2C4H7
C18H33O2C2H3
66
Octadecenyl acetateg
C18H35O2C2H3
310 250
67
68
69
70
71
72
73
74
75
76
77
78
Hexadecyl butyrate
Octadecyl acetate
Unidentified
Heptadecadienyl butyrate1 C17H33O2C4H7
C
C
16H33O2C4H7
18H37O2C2H3
312 224
312 252
308 308, 135, 121, 81, 67, 41 31.17
322 234
324 236
31.35
31.37
31.65
31.66
31.73
32.00
32.00
32.37
32.64
32.72
32.88
32.88
32.98
33.23
34.18
Heptadecenyl butyrateb
Pentadecyl caproate
Nonadecenyl acetateb
Heptadecyl butyrate
Eicosenyl formateb
Nonadecyl acetate
C17H33O2C4H7
C15H31O2C6H11 328 177, 210
C19H37O2C2H3
C17H35O2C4H7
C20H39OCHO
324 264
326 238
324 278
326 266
336 248
338 250
C19H39O2C2H3
Octadecadienyl butyrateb C18H33O2C4H7
Octadecenyl butyrateh
C18H35O2C4H7
79
80
81
82
83
Eicosenyl acetateb
Hexadecyl caproate
Octadecyl butyrate
Eicosyl acetate
C20H39O2C2H3
338 278
C16H33O2C6H11 340 177, 224
C18H37O2C4H7
340 252
340 280
C20H41O2C2H3
Unidentified terpenoid
376, 333, 109, 69

CROCODILE VOLATILE COMPOUNDS
TABLE 1. CONTINUED
775
Molecular
formula
Characteristic ions
observed (m/z)
No.
Compound
NMM
Rt (min)
84
85
Hexadecyl caprylate
Octyl oleate
C16H33O2C8H15
C8H17
368
394
368, 224, 145
264
35.13
37.52
O2C18H33
C30H50
86
87
88
89
90
Squalene
410
368
450
386
414
41, 69, 81
368, 353, 147
264
386
368
38.09
39.06
40.70
42.54
43.95
Cholesta-3,5-diene
Dodecyl oleate
Cholesterol
C
C
27H44
12H25O2C18H33
C27H46
O
Cholesteryl formate
C27H45OCHO
a Compounds are numbered in order of increasing GC retention time. NMM = nominal molecular
mass. Trace components are not listed.
b Double bond position undetermined.
c Mixture of (Z)-hexadec-7-en-1-yl acetate (major) and (Z)-hexadec-9-en-1-yl acetate (minor).
d Mixture of (Z)-octadec-9-en-1-ol (major) and (Z)-octadec-11-en-1-ol (minor).
e Mixture of (Z)-heptadec-8-en-1-yl acetate (major) and (Z)-heptadec-9-en-1-yl acetate (minor).
f
Mixture of (Z)-octadec-9-en-1-yl formate (major) and (Z)-octadec-11-en-1-yl formate (minor).
g Mixture of (Z)-octadec-9-en-1-yl acetate (major) and (Z)-octadec-11-en-1-yl acetate (minor).
h Mixture of (Z)-octadec-9-en-1-yl butyrate (major) and (Z)-octadec-11-en-1-yl butyrate (minor).
the Chinese alligator (A. sinensis) (Dunn et al., 1993) and caimans (Paleosuchus
spp.)(Shafagatietal., 1989), althoughcitronellolhasbeendescribedonlyfromsev-
eral caimans. 2,3-Dihydrofarnesyl acetate, a known paracloacal gland component
of the brown caiman (Caiman crocodilus fuscus) (Wheeler et al., 1999), accounted
for up to 4% of the secretion of immature crocodiles, but was not detected in the
adult samples.
Interestingly, we did not find free formic, acetic, or butyric acids in any of
the secretions, based on the derivatization of the total sample with pentafluoroben-
zyl bromide/triethylamine (Attygalle, 1998). Five C14–C18 fatty acids, which are
routinely encountered from vertebrate integumentary organs, were observed in im-
mature C. acutus, and a significant amount of oleic acid ( 5%) was found in the
adult male; we failed to detect free fatty acids in hatchling C. acutus. Thirteen fatty
acids ranging from C14 to C20 were reported from the skin glands of Crocodylus
spp. by Navajas Polo et al. (1988a,b). It should be noted that these investiga-
tors combined, for their studies, the paracloacal and gular (throat) glands of both
C. acutus and the Cuban crocodile (C. rhombifer) thus our results are not directly
comparable with theirs. We were also unable to confirm the presence of an in-
triguing lactone related to 10-hydroxystearic acid described in an earlier report
(Navajas Polo, 1982) from the macerated and steam-distilled skin glands of the
aforementioned mixed species.
Squalene was the major hydrocarbon we detected in C. acutus. This com-
pound was especially abundant in the adult female; it was either present in reduced

´
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
776
TABLE 2. RELATIVE AMOUNTS OF COMPOUNDS IDENTIFIED FROM PARACLOACAL GLANDS
OF HATCHLING, IMMATURE, AND ADULT Crocodylus acutusa
Immatures
Adults
Compound
Hatchlings (5) Female (5) Male (6) Female (1) Male (1)
Alkyl and alkenyl esters
Dodecyl formate
Tridecyl formate
<0.2
1.30
<0.2
2.07
1.10
8.16
1.30
4.44
ND
4.12
0.59
2.31
0.99
ND
<0.2
1.02
<0.2
<0.2
<0.2
<0.2
0.24
<0.2
1.09
0.57
1.53
ND
2.53
<0.2
<0.2
0.32
<0.2
0.73
0.31
2.91
1.83
4.92
ND
ND
ND
ND
ND
ND
0.51
0.96
ND
ND
ND
<0.2
Tetradecenyl formate
Tetradecyl formate
Pentadecenyl formate
Pentadecyl formate
7-Hexadecenyl formate
Hexadecyl formate
Heptadecadienyl formate
Heptadecenyl formate
Heptadecyl formate
Octadecenyl formate
Octadecyl formate
Eicosenyl formate
0.63
0.30
2.50
2.07
6.89
ND
9.26
0.89
20.06
4.80
<0.2
ND
ND
ND
<0.2
ND
<0.2
0.77
0.91
11.28
2.62
<0.2
<0.2
0.31
<0.2
1.14
<0.2
0.71
3.28
<0.2
1.72
9.47
1.79
8.90
0.84
2.19
13.84
4.29
<0.2
<0.2
<0.2
<0.2
0.23
<0.2
0.54
<0.2
0.74
0.28
2.09
<0.2
<0.2
ND
1.67
1.39
ND
ND
ND
ND
3.73
0.61
<0.2
<0.2
0.45
<0.2
1.65
<0.2
0.64
3.48
<0.2
1.21
10.85
2.02
6.13
0.79
4.10
14.40
3.73
<0.2
<0.2
<0.2
<0.2
<0.2
ND
Dodecyl acetate
Tridecyl acetate
Tetradecenyl acetate
Tetradecyl acetate
3.82
ND
1.90
7.25
<0.2
Pentadecadienyl acetate
Pentadecenyl acetate
Pentadecyl acetate
Hexadecadienyl acetate
Hexadecenyl acetate
Hexadecyl acetate
Heptadecadienyl acetate
Heptadecenyl acetate
Heptadecyl acetate
Octadecadienyl acetate
Octadecenyl acetate
Octadecyl acetate
Nonadecenyl acetate
Nonadecyl acetate
Eicosenyl acetate
Eicosyl acetate
Tridecyl butyrate
Tetradecenyl butyrate
Tetradecyl butyrate
Pentadecenyl butyrate
Pentadecyl butyrate
Hexadecenyl butyrate
Hexadecyl butyrate
Heptadecadienyl butyrate
ND
ND
ND
ND
0.97
4.04
ND
2.51
1.67
ND
7.03
4.58
ND
ND
ND
ND
ND
ND
ND
0.86
<0.2
ND
0.61
3.59
ND
4.31
0.34
6.09
9.90
2.28
ND
ND
ND
ND
ND
ND
2.06
19.97
1.02
5.36
0.73
1.15
9.10
3.30
ND
ND
<0.2
<0.2
<0.2
ND
0.32
<0.2
0.60
<0.2
1.98
<0.2
0.36
ND
0.93
<0.2
1.11
<0.2
<0.2
<0.2
ND
0.45
0.76
0.76
ND
ND
ND
ND
2.25
ND

CROCODILE VOLATILE COMPOUNDS
TABLE 2. CONTINUED
777
Immatures
Adults
Compound
Hatchlings (5) Female (5) Male (6) Female (1) Male (1)
Heptadecenyl butyrate
Heptadecyl butyrate
Octadecadienyl butyrate
Octadecenyl butyrate
Octadecyl butyrate
Pentadecyl caproate
Hexadecyl caproate
Hexadecyl caprylate
Methyl palmitate
Octyl oleate
0.422
<0.2
ND
0.61
0.20
<0.2
<0.2
ND
ND
ND
ND
0.33
<0.2
<0.2
0.64
0.40
<0.2
<0.2
<0.2
<0.2
ND
0.69
<0.2
<0.2
0.98
<0.2
ND
<0.2
ND
<0.2
<0.2
<0.2
ND
ND
ND
<0.2
<0.2
ND
ND
ND
ND
ND
0.88
0.42
ND
3.30
0.94
ND
ND
ND
ND
ND
ND
Dodecyl oleate
<0.2
ND
Terpenoid Esters
Citronellyl formate
Citronellyl acetate
Citronellyl butyrate
Dihydrofarnesyl acetate
Alcohols
0.26
1.05
0.66
4.75
ND
ND
ND
0.63
ND
ND
<0.2
ND
ND
ND
ND
ND
ND
ND
0.41
<0.2
Tridecyl alcohol
Tetradecyl alcohol
Pentadecyl alcohol
Hexadecenyl alcohol
Hexadecyl alcohol
Heptadecadienyl alcohol
Heptadecenyl alcohol
Heptadecyl alcohol
Octadecenyl alcohol
Octadecyl alcohol
Acids
ND
ND
0.23
ND
0.58
ND
<0.2
ND
<0.2
0.23
1.16
<0.2
2.30
<0.2
0.26
<0.2
1.00
0.37
ND
<0.2
ND
ND
ND
ND
ND
0.22
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.68
<0.2
1.24
0.26
1.23
<0.2
2.30
<0.2
0.26
ND
ND
ND
ND
0.46
ND
Myristic acid
Pentadecanoic acid
Palmitic acid
Oleic acid
Stearic acid
ND
ND
ND
ND
ND
<0.2
<0.2
<0.2
0.22
<0.2
<0.2
0.35
0.88
<0.2
ND
ND
ND
ND
ND
ND
ND
ND
4.89
ND
<0.2
Hydrocarbons
Pentadecane
Squalene
ND
ND
<0.2
3.44
ND
0.74
ND
52.00
ND
0.33
Steroids
Cholesta-3,5-diene
Cholesterol
ND
ND
ND
<0.2
0.27
<0.2
<0.2
<0.2
<0.2
ND
ND
ND
ND
<0.2
ND
Cholesteryl formate
Unidentified compounds
Terpenoid No. 4
Terpenoid No. 10
Terpenoid No. 12
<0.2
<0.2
<0.2
0.45
<0.2
<0.2
<0.2
<0.2
<0.2
<0.2
ND
ND
ND
ND
ND

´
778
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
TABLE 2. CONTINUED
Immatures
Adults
Female (1)
Compound
Hatchlings (5)
Female (5)
Male (6)
Male (1)
Terpenoid No. 15
Terpenoid No. 18
Unidentified No. 21
Unidentified No. 32
Unidentified No. 42
Unidentified No. 47
Unidentified No. 53
Unidentified No. 58
Unidentified No. 69
Terpenoid No. 83
1.37
ND
ND
0.25
1.50
0.78
<0.2
<0.2
<0.2
<0.2
0.27
ND
<0.2
<0.2
<0.2
0.71
<0.2
0.54
0.52
<0.2
<0.2
0.27
<0.2
1.61
ND
0.77
<0.2
<0.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.46
ND
3.00
ND
ND
<0.2
ND
a Numbers in parentheses indicate the number of individuals sampled. ND = non detected.
FIG. 2. Distribution of the alkyl and alkenyl esters (formates, 9–62; acetates, 14–68; and
butyrates, 31–81) found in average abundance >0.2% in the paracloacal gland secretion of
Crocodylus acutus, expressed as a percentage of the total secretion. Percentages are listed
in Table 2; numbers refer to components listed in Table 1. Crocodile hatchlings were not
sexed.

CROCODILE VOLATILE COMPOUNDS
779
quantitiesorwasentirelyabsentinthemaleorimmaturefemales. Theabundanceof
squalene in our adult female is consistent with the appearance of an intense band
corresponding to hydrocarbons in the thin-layer chromatogram of a female C. acu-
tus secretion sample examined in the earlier survey of paracloacal gland lipids by
Weldon and Tanner (1991).
All our compound identifications are based on the mass spectra obtained
by GC-MS and comparison with synthetic standards. In addition, the identifi-
cation of the major components was supported by infrared spectra obtained by
gas chromatography–Fourier transform IR spectroscopy. Gas-phase FTIR spec-
tra proved particularly helpful in recognizing formate, acetate, and butyrate peaks.
Thus, the carbonyl group stretching vibrations appeared characteristically at 1740–
1
1748 cm in the gas-phase FTIR spectra of C13–C18 formates, whereas that of
acetates and butyrates appeared at 1761–1762 and 1753–1754 cm 1, respectively.
FTIR spectra were also useful for the assignment of the Z configuration to the
double bonds in the unsaturated esters and alcohols. Double-bond positions in 9-
and 11-octadecenyl acetate, 8- and 10-heptadecenyl acetate, 7- and 9-hexadecenyl
acetate, 9-octadecenyl formate, 8-heptadecenyl formate, and 7-hexadecenyl for-
mate were determined by the DMDS derivatization method (Buser et al., 1983;
Francis and Veland, 1981).
We did not observe in C. acutus either dianeackerone or the related steroidal
esters characteristic of the confamilial Osteolaemus tetraspis (Whyte et al., 1999;
Yang et al., 1999); conversely, neither the aliphatic esters, alcohols, nor squalene
that we have observed in C. acutus have been found in O. tetraspis. These re-
sults, foreshadowed by earlier TLC studies (Weldon and Tanner, 1991; Weldon
and Wheeler, 2000), demonstrate substantive chemical differences in the paracloa-
cal gland products of Crocodylus and Osteolaemus. While further analyses of the
glandular products of Crocodylus will be needed to assess the range of chemical
variation within the Crocodylidae, we can anticipate that the deeper task of de-
ciphering the biological significance of these chemically complex secretions will
remain a challenge for some time to come.
Acknowledgments—We are grateful to K. Bartel, B. Schwedick (Cypress Gardens, Plant City,
Florida), A. Register, P. Register (Gatorama, Palmdale, Florida), J. Brueggen, J. Darlington, J. Heil,
S. Huber, D. Kledzik, W. Puckett, and T. Rexroad (St. Augustine Alligator Farm, Florida) for allowing
access to animals in their collections. The partial support of this research by a grant from the Research
Corporation (DS0260), as well as a postdoctoral fellowship from the Pew Latin American Fellows
Program in the Biomedical Sciences (P0236SC) to S.G.R., is acknowledged with pleasure.
REFERENCES
ATTYGALLE, A. B. 1998. Microchemical techniques, pp. 270–271, in J. G. Millar and K. F. Haynes
(eds.). Methods in Chemical Ecology, Vol. 1: Chemical Methods. Chapman & Hall, Norwell,
Massachusetts.

´
GARCIA-RUBIO, ATTYGALLE, WELDON, AND MEINWALD
780
ATTYGALLE, A. B., and MORGAN, E. D. 1986. A versatile micro-reactor and extractor. Anal. Chem.
58:3054–3058.
ATTYGALLE, A. B., MEINWALD, J., and EISNER, T. 1992. Defensive secretion of a carabid beetle
Hellouomorphoides clairvillei. J. Chem. Ecol. 18:489–498.
ATTYGALLE, A. B., SMEDLEY, S., MEINWALD, J., and EISNER, T. 1993. Defensive secretion of two
notodontid caterpillars (Schizura unicornis, S. badia). J. Chem. Ecol. 19:2089–2104.
BURGER, B. V., LE ROUX, M., SPIES, H. S. C., TRUTER, V., BIGALKE, R. C., and NOVELLIE, P. A.
1981. Mammalian pheromone studies, V. Compounds from the preorbital gland of the grysbok,
Raphicerus melanotis. Z. Naturforsch. 36c:344–346.
BURGER, B. V., TIEN, F.-C., LE ROUX, M., and MO, W.-P. 1996. Mammalian exocrine secretions: X.
Constituents of preorbital secretion of grysbok, Raphicerus melanotis. J. Chem. Ecol. 22:739–764.
BURGER, B. V., GREYLING, J., and SPIES, H. S. C. 1999. Mammalian exocrine secretions. XIV. Con-
stituents of preorbital secretion of steenbok, Raphicerus campestris. J. Chem. Ecol. 25:2099–2108.
BUSER, H.-R., ARN, H., GUERIN, P., and RAUSCHER, S. 1983. Determination of double bond position
in monounsaturated acetates by mass spectrometry of dimethyl disulfide adducts. Anal. Chem.
55:818–822.
DUNN, B. S., JR., WELDON, P. J., HOWARD, R. W., and MCDANIEL, C. A. 1993. Lipids from the
paracloacal glands of the Chinese alligator (Alligator sinensis). Lipids 28:75–78.
EISNER, T., and MEINWALD, J. 1995. Chemical Ecology: The Chemistry of Biotic Interaction. National
Academy Press, Washington, D.C.
FRANCIS, G. W., and VELAND, K. 1981. Alkylthiolation for the determination of double bond positions
in linear alkenes. J. Chromatogr. 219:379–384.
IBRAHIM, S. A., AVERY, J. W., WELDON, P. J., and WHEELER, J. W. 1998. Age-class differences in lipids
from the paracloacal glands of the American alligator (Alligator mississippiensis). Z. Naturforsch.
53c:201–209.
IHARA, M., YASUI, K., TANIGUCHI, N., and FUKUMOTO, K. 1990. Asymmetric total synthesis of ( )-
protoemetinol , ( )-protoemetine, ( )-emetine and ( )-tubolosine by highly stereocontrolled
radical cyclizations. J. Chem. Soc. Perkin Trans. 1:1469–1476.
KAPLAY, K. M., SAHNI, R., DAMORADAN, N. P., and DEV, S. 1980. Photochemical transformations-II.
Organic iodides II: Citronellyl iodide, 2,3-dihydro-6(Z)-farnesyl and 2,3-dihydro-6(E)-farnesyl
iodide. Tetrahedron 36:1455–1461.
KUSHLAN, J. A., and MAZZOTTI, F. J. 1989. Population biology of the American crocodile. J. Herpetol.
23:7–21.
LOPEZ, L. C., MORGAN, E. D., and BRAND, J. M. 1993. Hexadecanol and hexadecyl formate in the
venom gland of formicine ants, Phil. Trans. R. Soc. London B. 341:177–180.
MADDEN, B. A., and PRESTWICH, G. D. 1994. Asymmetric synthesis of a mechanism-based inhibitor
of oxidosqualene cyclase. J. Org. Chem. 59:5488–5491.
MO, W.-P., BURGER, B. V., LE ROUX, M., and SPIES, H. S. C. 1995. Mammalian exocrine secretions:
IX. Constituents of preorbital secretion of oribi, Ourebia ourebi. J. Chem. Ecol. 21:1191–1215.
NAVAJAS POLO, C. 1982. Procedimiento para la obtencio´n de una lactona y el producto obtenido.
Cuban patent #21412.
NAVAJAS POLO, C., ROSADO, A., MAGRANER, J., and RODRIGUEZ, G. 1988a. Estudio quimico del almiz-
cle del genero Crocodylus que habita en Cuba. I-A. Algunos acidos grasos saturados presentes.
Rev. Cubana Farm. 22:61–68.
NAVAJAS POLO, C., ROSADO, A., MAGRANER, J., and RODRIGUEZ, G. 1988b. Estudio quimico del
almizcle del genero Crocodylus que habita en Cuba. I-B. Algunos acidos grasos insaturados
presentes. Rev. Cubana Farm. 22:69–75.
SHAFAGATI, A., WELDON, P. J., and WHEELER, J. W. 1989. Lipids in the paracloacal gland secretions
of dwarf (Paleosuchus palpebrosus) and smooth-fronted (P. trigonatus) caimans . Biochem. Syst.
Ecol. 17:431–435.

CROCODILE VOLATILE COMPOUNDS
781
WELDON, P. J., and FERGUSON M. J. 1993. Chemoreception in crocodilians: Anatomy, natural history,
and empirical results. Brain Behav. Evol. 41:239–245.
WELDON, P. J., and TANNER, M. J. 1991. Gular and paracloacal gland secretions of crocodilians: A
comparative analysis by thin-layer chromatography. Biochem. Syst. Ecol. 19:133–141.
WELDON, P. J., and WHEELER, J. W. 2000. The chemistry of crocodilian skin glands, pp. 286–296, in
G. C. Grigg, F. Seebacher, and C. E. Franklin (eds.). Crocodilian Biology and Evolution, Surrey
Beatty & Sons, Chipping Norton, New South Wales, Australia.
WHEELER, J. W., IBRAHIM, S. A., and WELDON, P. J. 1999. 2,3-Dihydrofarnesyl and citronellyl esters
in the paracloacal gland secretions of the brown caiman (Caiman crocodilus fuscus) from Costa
Rica. Biochem. Syst. Ecol. 27:27–32.
WHYTE, A., YANG, Z., WELDON, P. J., EISNER, T., and MEINWALD, J. 1999. Reptilian chemistry:
Characterization of dianeackerone, a secretory product from a crocodile. Proc. Natl. Acad. Sci.
USA 96:12246–12250.
YANG, Z., WHYTE, A., ATTYGALLE, A. B., WELDON, P. J., EISNER, T., and MEINWALD, J. 1999.
Reptilian chemistry: Characterization of a family of dianeackerone-related steroidal esters from
a crocodile secretion. Proc. Natl. Acad. Sci. USA 96:12251–12256.
YANG, Z., ATTYGALLE, A. B., and MEINWALD, J. 2000. Reptilian chemistry: Enantioselective syntheses
of novel components from a crocodile exocrine secretion. Synthesis 13:1936–1943.
ZHDANOV, R. I., and ZHENODAROVA, S. M. 1975. Chemical methods in oligonucleotide synthesis.
Synthesis 222–245.