Barbara French
PO
Box 162603
Austin,
Texas 78716
(512)
327-9721
(512)
327-9724 Fax
french@batcon.org
identification of characteristic odors of Tadarida
brasiliensis mexicana by AromaTraxä
Gas Chromatography/olfactometry
Lawrence T. Nielsen, David K. Eaton, and Donald W.
Wright,
Barbara A. French,
Bat Conservation
International, Austin, TX 78746
Abstract-
The odors in a central Texas cave with a large roosting population of
Mexican free-tailed bats (Tadarida brasiliensis mexicana) were identified
and related to captive individual bats. Solid
phase microextraction (SPME) was used to sample and concentrate the volatile
organics from the cave and individual bats.
Odors were detected organoleptically and simultaneously quantified and
identified. The characteristic odor for T. b. mexicana is due principally
to 2’-aminoacetophenone.
Resumen-
Se identificaron los olores en una cueva de Texas Central con una gran
población de murciélago mexicano de cola libre (Tadarida brasiliensis
mexicana) y fueron relacionados con los procedentes de individuos cautivos.
Para extraer y concentrar los compuestos orgánicos volátiles, tanto de
la cueva como de los ejemplares cautivos, se utilizó la técnica de
microextracción en fase sólida (MEFS). Los
olores se detectaron organolépticamente y fueron identificados y cuantificados
de forma simultánea. El olor característico de T.
b. mexicana se debe principalmente a la 2'-aminoacetofenona.
Introduction-
Bats use chemical cues for food detection, social communication, kin
recognition, and individual or group identification (Gustin and McCracken, 1987;
Suthers, 1970; Loughry and McCracken, 1991; De Fanis and Jones, 1995; Bloss,
1999; Bouchard, 2001). Some bats exhibit a preference for odors from individuals
originating from their own colonies, and both sex discrimination and roostmate
recognition have been associated with use of olfactory cues (Bouchard, 1999; De
Fanis and Jones, 1995; Bloss, et al. 2002).
Odor-producing organs are present in many species and appear to play an
important role in courtship display and mate selection (Voigt and von Helversen,
1999; Voight, 2002).
The roosts of the Mexican free-tailed bat (Tadarida brasiliensis mexicana) are known for their characteristic odor. Females of this
species use chemical cues to identify their young among millions of pups, and males can discriminate their own odors from those of other
males (Gustin and McCracken, 1987). A characteristic odor can be sensed at a considerable distance from a roost and has a distinctive
”corn tortilla” or ”taco shell” aroma. The odor is stronger closer to the roost and not described as ”taco shell” at the elevated concentrations
encountered there. The odor character inside the cave is more complex than that of the ”taco shell” aroma alone.
To identify the characteristic odors of this species, the volatile compounds from Bracken Cave, a Mexican free-tailed roost in central
Texas, were collected by solid phase microextraction (SPME) and analyzed by gas chromatrography-olfactometry (GC-O). Odors from
captive, hand-fed bats were then collected and analyzed in the same way for comparison.
Methods
and Materials-
Sampling of the volatile organic compounds in the Bracken Cave
environment was facilitated by the existence of an artificial ventilating shaft
from the interior that had a continuous draft of air.
SPME fibers (Carboxen/PDMS, 85 micron, 2 cm length, 23 gauge, on
StableflexÔ
Supelco, Supelco Park, Bellefonte, PA, 16823-0048) were suspended into the air
flow. Five sample collections were
made for 120 minutes each on 30 June 2001 and 4 collections were made on 31
August 2001. After sampling, the
fibers were wrapped in conditioned aluminum foil and analyzed within 1-2 days
after collection.
Sampling of fabric roosting pouches of 5 captive bats (T. brasiliensis)
originating from central Texas took place in 2001 on 7 September (2 roosts), 24
September (1 roost), and 12 October (2 roosts).
Samples were collected by inserting an SPME fiber into each cloth
roosting pouch for various lengths of time.
The cloth pouches were used by only one individual but were open to the
ambient air. Unused pouches were
also sampled and analyzed as blanks.
Urine samples were collected from captive bats also originating from
central Texas. Samples were
collected from 3 bats (T. brasiliensis) on 16 September 2001 and from 5
bats (T. brasiliensis) on 30 September 2001. For comparison purposes,
urine samples were also collected from a female Lasiurus cinereus on 30
October, a female Lasiurus intermedius on 31 October, a male Nycticeius
humeralis on 30 October, and a male Myotis velifer on 30 October.
Urine was collected from bats with glass pipettes and placed in 40 ml
Eagle-Picher EPA vials. Glass
pipettes were also touched to the gular glands of 2 captive males (T.
brasiliensis) and to the anus of 1 captive male (T. brasiliensis) on
16 September 2001 and placed in EPA vials.
The SPME fibers were then inserted into the vials through the vial septa
and exposed to the urine and glandular volatiles for various lengths of time.
Odor
analysis was done on a standard configuration AromaTraxÔ
instrument (Microanalytics, Round Rock, TX).
The inlet for the thermal desorption of the SPME fibers was equipped with
a Merlin MicrosealÔ
septum. Odor volatiles were
separated on the AromaTraxÔ
system using the standard arrangement of tandem BP1 and BP20 columns and
detected simultaneously with photoionization (PID), mass spectral (MS) and
olfactory detectors. The sniff port
olfactory response was recorded using AromaTraxÔ
odor tracking software.
To identify the hundreds of volatiles in the Bracken cave samples, it was
necessary to use the multidimensional gas chromatography (MDGC) capability of
the AromaTraxÔ
system to enhance separation and identification of individual odor compounds.
Identification of odor compounds was made by use of BenchTop/PBM Software
Library Search program (Palisade Corp., N. Y.).
Simultaneous detection of the resolved odors was done using PID, MS and
olfactory detection.
Results-
Cave Environment-
Bracken Cave was occupied by an estimated 20 million Mexican free-tailed bats on the two sampling dates and gave essentially the
same odor compositional results on both dates. Hundreds of volatile compounds were detected; the 76 most concentrated compounds
are listed in Table 1. The 120 minute SPME collection reveals 2’-aminoacetophenone as the most concentrated compound in the cave
environment. It is also the most intense odor sensed at the sniff port during GC-O analysis and the most characteristic of the cave
environment. The next most intense odors are the earthy odor of 1-octen-3-one, the phenolic odor of 2-chlorophenol, and the floral or
herbaceous aroma of what is tentatively identified as 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one.
Captive Individuals
Occupied roosting pouches of 5 T. brasiliensis were sampled and
GC-O results corrected for odors common to unused pouches.
The ”taco shell” aroma of 2’-aminoacetophenone was found for all 5
individuals (Table 2). One male had
only 2 detectable odors whereas the other bats had 7 to 12 odors.
Five of the 19 odors from individual profiles were among the major odors
from Bracken Cave occupied by free-ranging bats including octanal, acetic acid,
isovaleric acid, 4-(2,6,6-trimethyl-1-cyclohexenyl)-3-buten-2-one, and
2-aminoacetophenone (Table 1).
All
7 T. brasilensis bats had the characteristic ”taco shell” odor of
2’-aminoacetophenone in their urine (Table 3).
Except for acetic acid and butyric acid detected in most samples, there
was considerable variation of other odor compounds among the 7 bats’ urine.
Ten of the odors found in urine samples were also found in the roosting
pouches.
The odor of 2’- aminoacetophenone was not found in the urine of Lasiurus
cinereus, Lasiurus intermedius, Nycticeius humeralis, or Myotis
velifer individuals (Table 4). Lasiurus cinereus had a strong
characteristic amine odor identified as trimethylamine.
No single strong characteristic odor was detected from Lasiurus
intermedius, Nycticeius humeralis, or Myotis velifer
individuals.
Fluid from the gular gland of 1 male (T. brasiliensis) had only
acetic acid and another somewhat sour odor.
Fluid from a second male (T. brasiliensis) had sour acetic and
propionic acids, a nutty pyrazine odor, and 2’-aminoacetophenone.
Other odors were detected in the analysis, but they were also present in
the unused roosting pouch material.
Discussion and Conclusions-
Collection of volatile compounds by solid phase microextraction and
analysis with Aromatraxä
gas chromatography/olfactometry was successful in identifying
2’-aminoacetophenone as the odorant responsible for the characterisitic odor
of Tadarida brasiliensis mexicana. The
chemical was identified in Bracken Cave ambient air and from individual captive
bats of this species.
Preliminary
data from this study suggest that there are measurable odor differences between
individual bats of this species. French
and Lollar (1998) have noticed distinct differences between individuals in
captive colonies, particularly during scent marking activity by males.
In this study, there was much variation in urine odors from captive bats.
However, only single samples were analyzed from individual bats.
Dietary differences between free-ranging bats and captives fed lesser
mealworms (Tenebrio molitor) might also result in differing odor
profiles.
We had difficulty in assuring that fluid from the gular gland was actually being sampled due to the minute amount of secretion available. This could indicate that the method was not sufficiently reproducible to consistently detect low intensity odors. Optimization of sampling techniques and further investigation of the reproducibility of volatile compound and odor profiles is needed to determine if this system is valid for investigating odor discrimination among individual bats.
The technology presented in this paper may be of value in qualifying bat
behaviors associated with familial relationships, mate selection,
or
parturition. It could also be
useful in the study of multiple species’ use of a roost, in absentia species
identification at roost sites, or
even the development of attractants for bats
that might enhance artificial habitat use.
Ultimately, the AromaTraxä
gas
chromatography/olfactometry system may prove of value in further
investigation of many aspects of the chemical ecology of bats.
Literature
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