Novel chemistry of abdominal defensive glands of nymphalid butterfly Agraulis vanillae

Article abstract of DOI:10.1023/A:1010372114144

Abdominal defensive glands of both sexes of the Gulf fritillary butterfly, Agraulis vanillae (Linnaeus) (Nymphalidae:Heliconiinae) emit a pronounced odor when disturbed. We have identified 6-methyl-5-hepten-2-one; oleic, palmitic, and stearic esters of the corresponding alcohol 6-methyl-5-hepten-2-ol; hexadecyl acetate; 1,16-hexadecanediol diacetate; and 1,15-hexadecanediol diacetate in the glandular exudate. Since we have determined that free-flying birds or birds in a butterfly conservatory discriminate against A. vanillae as prey, we suggest that the constituents in the glands may play a defensive role against potential avian predators.

Full text of DOI:10.1023/A:1010372114144

Journal of Chemical Ecology, Vol. 27, No. 6, 2001
NOVEL CHEMISTRY OF ABDOMINAL DEFENSIVE
GLANDS OF NYMPHALID BUTTERFLY Agraulis vanillae
GARY N. ROSS,1 HENRY M. FALES,2, HELEN A. LLOYD,2,5 TAPPEY
JONES,3 EDWARD A. SOKOLOSKI,2 KIMBERLY MARSHALL-BATTY,2
and MURRAY S. BLUM4
16095 Stratford Avenue, Baton Rouge, Louisiana 70808
2Laboratory of Biophysical Chemistry, NHLBI, NIH
Bethesda, Maryland 20892
3Department of Chemistry
Virginia Military Institute
Lexington, Virginia 24450
4Department of Entomology
University of Georgia
Athens, Georgia 30602
5Present address: Sequim, Washington 98382
(Received October 5, 2000; accepted February 12, 2001)
Abstract—Abdominal defensive glands of both sexes of the Gulf fritillary
butterfly, Agraulis vanillae (Linnaeus) (Nymphalidae:Heliconiinae) emit a pro-
nounced odor when disturbed. We have identified 6-methyl-5-hepten-2-one;
oleic, palmitic, and stearic esters of the corresponding alcohol 6-methyl-5-
hepten-2-ol;hexadecylacetate;1,16-hexadecanedioldiacetate;and1,15-hexade-
canediol diacetate in the glandular exudate. Since we have determined that free-
flying birds or birds in a butterfly conservatory discriminate against A. vanillae
as prey, we suggest that the constituents in the glands may play a defensive role
against potential avian predators.
Key Words—Agraulis vanillae, defensive glands, 6-methyl-5-hepten-2-one,
6-methyl-5-hepten-2-ol esters, 1,15-hexadecanediol diacetate 1,16-hexadecane-
diol diacetate.
INTRODUCTION
The Gulf fritillary butterfly, Agraulis vanillae (Nymphalidae), was first described
by Linnaeus (1758), who characterized it as a species whose brilliant coloration
To whom correspondence should be addressed.
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made it very conspicuous. Agraulis is the most primitive genus in the large neotrop-
ical subfamily Heliconiinae, a taxon that is famous for the development of mimicry
theory utilizing both brightly colored unpalatable and palatable species of butter-
flies (Mu¨ller, 1886; Ross, 1995). This generally tropical butterfly is also unusual in
its marked northern extension that reaches to Georgia, the Carolinas, and southern
Missouri and Nebraska (Glassberg, 1999) in the United States (Emsley, 1965). The
wingpatternisknownforitsstrikingcontrastcolorationwithsilverbelowthewings
and triangles and polygons of alternating silver, yellow, red, and brown above.
Tyrosine is responsible for the black and brown colors via melanin formation, while
tryptophan forms the red, light brown, and tan portions via 3-hydroxykynurenine
and ommochromes as shown by incorporation of these radiolabeled precursors
(Nijhout and Koch, 1991).
Eltringham (1925) has discussed the observation of Mu¨ller (1886) on the
emission of a “strong and nauseous smell” emanating from glands in the anal valvu-
lae of the males and from a yellow gland extruded on the abdominal dorsum of the
females of A. vanillae. Mu¨ller (1886) called this species the Maracuja butterfly be-
cause the larvae generally fed on the maracuja (Passiflora edulis, Passifloriaceae),
also known as the passion flower, on which it does considerable damage. The
odor of the exudate from the females was said to resemble that of phenylcarby-
lamine (phenylisocyanide) (Eltringham, 1925), but then again “not unpleasant”
and “more complex than phenylcarbamylamine.” We report below the chemical
nature of some of the compounds in the exudates of male and female A. vanillae.
The above workers all described the repellent functions of these odorous secre-
tions, and we have made additional observations with predatory birds in view of
the possible unpalatability of both females and males of A. vanillae.
METHODS AND MATERIALS
Extraction and Subsequent Survival. Eight males and five females were col-
lected from a personal butterfly garden belonging to Ross (1999) in Baton Rouge,
Louisiana on July 22, 1999. The butterflies were initially placed individually in
glassine envelopes for temporary storage. After being brought inside, each but-
terfly was handheld with pressure exerted on the thorax and upper abdomen. The
pressure forced extrusion of the glands, which were then punctured with a No.1
stainless steel insect pin. Squares of filter paper (7–8 mm) held with a watch-
maker’s pair of forceps were touched to the secretions for collection. The papers
were then stored in small glass vials, three for the females and two for the males,
each containing 1.0 ml of methyl alcohol. Separate pins were used for the males and
females. Forceps were cleaned first with soap and water and then with methyl al-
cohol between sampling from males and females. Following extraction (milking),
the butterflies were returned to their glassine envelopes, placed within lock-proof
sandwich bags (males and females separate), and then stored in a refrigerator until

GLAND CHEMISTRY OF NYMPHALID BUTTERFLY
1221
the following day. Milkings were done on four consecutive days; however, males
produced virtually no secretions on days 3 and 4, whereas females continued to
produce, albeit to a lesser degree, through day 4. By day 5, four of the males and
one of the females had died and milkings were discontinued.
The remaining eight live individuals were returned to the garden. Surprisingly,
after 15 min of resting in the summer sun, the butterflies began to fly, feebly at first,
but in time, more naturally. Since the garden is full of flowers, including lantana, a
favorite of Agraulis, most of the released butterflies began to feed. When checked
3 hr later, three males and three females were still present and flying about with
no apparent difficulties. The following day only one male and two females were
seen. The females were flying about a Passiflora vine attempting to oviposit, but
the milking procedures had apparently damaged some critical component of their
reproductive tracts since no deposition of eggs could be observed. Two females
continued to remain within the garden for a total of four days.
Chemical Analysis. Mass spectra were run using an LKB 2091 mass spec-
trometer operating in the EI mode with a 15-m 0.25-mm DB-1 column with
0.25- m film thickness operating from 78 to 300 C at 8 C/min, with a helium
flow rate of 10 ml/min. Samples (1 l) of the extracts dissolved in methanol were
injected at 250 C using a splitless injector.
Samples of 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol were
obtained from Aldrich (Milwaukee, Wisconsin). Hexadecyl acetate and 1,16-
hexadecanediol diacetate were prepared by reacting equimolar amounts of the
corresponding alcohols (Aldrich) with an excess of acetic anhydride in pyridine
overnight. Evaporationoftheexcessreagentsandsolventleftthecrudeacetatesthat
were used without further purification. The 1,2- and 2,3-hexadecanediol acetates
were similarly prepared from samples of the diols prepared from the correspond-
ing hydrocarbons in conjunction with elucidation of the structures of diols found
in chicken uropygial glands (Haahti and Fales, 1967). 1,3-Hexadecanediol diac-
etate was similarly prepared by acetylation of the corresponding diol prepared by
treating commercial tetradecanal from Fluka (Milwaukee, Wisconsin) with com-
mercial vinylmagnesium bromide (Aldrich). The resulting crude 1-hexadecen-3-ol
was treated with an excess of borane dimethylsulfide complex (Aldrich). The usual
oxidative work-up with sodium hydroxide and hydrogen peroxide provided a sam-
ple of crude 1,3-hexadecanediol that was ca. 80% pure by gas chromatography.
1,15-Hexadecanediol diacetate was prepared by allowing an excess of 2.6
M methyllithium in ether (Aldrich) to react with 1 meq of ω-pentadecalactone
(Aldrich) dissolved in 20 ml of hexane. After standing 10 min, dilute hydrochloric
acid was added and the solution extracted with more ether. After drying with
˚
molecular sieves (3A, Fluka), the extracts were evaporated and a solid residue
obtained. This was easily recrystallized from 50% toluene in hexane, but NMR
revealed it to be a mixture. A portion of the sample was then separated on
100- m-thicksilicagelthinlayerchromatographyplates(Aldrich)with2%ethanol

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ROSS ET AL.
in chloroform. Three zones with retention factors (R f ) 0.63, 0.28, and 0.20 were
visualized with methyl red and their contents recovered by scraping and extraction
with 10% ethanol in chloroform. The zone at Rf = 0.63 was found by MS to be
recovered ω-pentadecalactone. The zone with R f = 0.28 melted at 45–62 C and
was found by GC-MS to be the desired 1-hexadecanol-15-one [m/z (%): 41(39),
43(100), 55(49), 67(14), 69(20), 71(29), 96(12), 180(3), 223(2), 226(1), 256(3,
M+)] contaminated with about 30% ethyl tetradecanoate, presumably from an
impurity of that acid in the lactone. Reduction of the keto alcohol with sodium
borohydride in ethanol overnight formed 1,15-hexadecanediol [m/z (%): 41(75),
43(60), 45(95), 55(100), 57(44), 67(40), 69(67), 71(28), 95(54), 96(41), 109(24),
111(25), 123(14), 124(15), 125(10), 137(8), 194(4), 196(6), 243(5), 258(0, M+)],
and acetylation with pyridine and acetic anhydride produced the diacetate, whose
mass spectrum and retention times were identical with the unknown diol diacetate
shown as peak 3 in Figure 2 below. The lower band of R f = 0.20 was found to
be the 2-methyl-1,15-hexadecanediol, mp 63–67 C, formed by secondary reac-
tion of the methyl ketone with excess methyllithium [m/z (%): 69(100), 71(55),
81(34), 83(78), 95(40), 97(45), 109(23), 111(18), 123(10), 125(8), 168(4), 180(3),
196(10), 221(4), 224(4), 239(9), 257(13, M-CH3), 272 (0, M+)].
Predatory Birds vs. Gulf Fritillary. Potential avian predators in the presence
of large populations of A. vanillae were observed in two situations. In the first,
50–70 butterflies were released weekly during 1996 into the “Butterflies in Flight”
exhibit at the Audubon Zoological Garden, New Orleans, Louisiana (the releases
were normal protocol for populating the conservatory). The exhibit included 60–70
free-flying birds, including aggressive insectivorous species such as red-rumped
caciques [Cacicus haemorrhous (Icteridae)] and superb starlings [Lamprotornis
superbus (Sturnidae)]. Avian predation on A. vanillae and other butterflies was
monitored almost daily for nine months by G. N. Ross as well as by zoo employees
and volunteers for approximately 60 hr, which included examination of the ground
for detached wings.
Avian predation was also monitored in an outdoor butterfly garden ( 350 m2)
in Baton Rouge, Louisiana, between 1997 and 1999 (Ross, 1999). A. vanillae,
whichutilizesPassifloraincarnataasahostplant(Ross1996, 2000b), wasthemost
common butterfly species by a factor of at least 10-fold. A variety of known avian
predators were present at all times in addition to a diversity of invertebrate species
with well-known predatory propensities. Efforts were made to directly observe A.
vanillae being utilized as a prey species or indirectly in terms of detached wings
being present on the ground.
RESULTS
Abdominal Glands and Their Secretions. The female possesses a pair of
posterior abdominal scent glands on the dorsum between abdominal segments 7

GLAND CHEMISTRY OF NYMPHALID BUTTERFLY
1223
and 8. These glands are bright yellow in color, spherical in shape (1.9–2.4 mm
in diameter), and appear spongy in texture. The paired structures are normally
inverted. However, when a butterfly is captured or disturbed, the glands are quickly
everted. Additionally, the dorsal membrane between abdominal segments 6 and 7
is partially extruded and appears bright yellow in color. If held within 15 cm, a
strong, very sweet odor is easily detected. If a gland is punctured with a fine pin, a
sizable quantity of bright yellow, clear fluid is emitted. This fluid has an acrid taste,
dries rapidly, and retains its color. The odor is not detectable after the fluid dries.
The male possessesa pairofbilobed, spherical structures(glands)1.4–1.8mm
in diameter within the valvae (claspers) of the abdominal tip. The structures appear
as bright yellow swollen pads when the claspers are extended, which occurs with
only the slightest provocation. The fragrance is more acrid and less sweet (more
alcoholic) than that produced by the females. If a gland is punctured, a small
amount (but less so than that of females) of clear yellowish fluid is released. The
fluid has the same fragrance as that emitted by an unaltered male. The taste is
acrid and similar to that of females, dries rapidly, and retains its color. The odor
dissipates on drying
Chemical Analysis. In spite of the subtle differences in odor described above,
samplesofthefluidobtainedfromeithermaleorfemaleabdominalglandsprovided
essentiallythesamegaschromatogramsandmassspectralanalyses. Figure1shows
a chromatogram of the male exudate where the early-eluting peak (peak 1) was
identified as 6-methyl-5-hepten-2-one via a search of the NIST-NIH-EPA database
FIG. 1. Gas chromatogram of a methanol extract of male abdominal glands of A. vanillae:
peak 1, 6-methyl-5-hepten-2-one; 2, hexadecyl acetate; 3, 1,15-pentadecanediol; 4, 1,16-
hexadecanediol diacetate; 5, palmitoyl ester of 6-methyl-5-hepten-2-ol; 6, oleoyl ester of
6-methyl-5-hepten-2-ol; 7, stearoyl ester of 6-methyl-5-hepten-2-ol.

1224
ROSS ET AL.
(NIST/NIH/EPA Mass Spectral Library, Standard Reference Data Program of the
National Institute of Standards and Technology, Gaithersburg, Maryland) and
by comparison of its mass spectrum and retention time with an authentic sam-
ple (Aldrich). Hexadecyl acetate (peak 2) was readily identified from its “acid
plus one” peak at m/z 61 and its M-60 ion at m/z 224, and its presence also
confirmed by comparison of its spectrum and retention time with an authen-
tic sample. Peak 3 is discussed in detail below. Peak 4 was identified as 1,16-
hexadecanediol diacetate through the database and by comparison with an authen-
tic sample.
Mass spectra of peaks 5, 6, and 7 all closely resembled each other with
abundant ions appearing at m/z 55, 69, 95, and 110 (Figure 2, peak 5). When
searched in the database, these ions produced the spectrum of 5-[(1,5-dimethyl-4-
hexenyl)oxy]-3-methyl-2-pentenoic acid ethyl ester where they were clearly due
to the (1,5-dimethyl-4-hexenyl)oxy- moiety. This fragment corresponds to the
alkoxy residue of 6-methyl-5-hepten-2-ol that is, in turn, the reduction product of
the ketone found in peak 1. Peaks 5, 6, and 7 then represent homologous long-chain
esters of that compound. Examination of minor peaks at higher mass identified
these as palmitic acid (m/z 256), oleic acid (m/z 282), and stearic acids (m/z 284),
respectively. To confirm the first of these assignments (peak 5), the palmitoyl ester
was prepared from 6-methyl-5-hepten-2-ol (Aldrich) and palmitoyl chloride in
pyridine. The product was indeed identical in retention time and mass spectrum
with that obtained from the insect in peak 5. These compounds do not appear to
have been encountered previously.
The mass spectrum of peak 3 resembled that of peak 4 (1,16-hexadecanediol
diacetate) in many respects, including the loss of two molecules of acetic acid to
form ions at m/z 222. However, peak 3 shows an ion at m/z 87 not seen in the 1,16-
diol, and this suggests the linkage CH3CH = OAc+. In fact, one of us (H.M.F)
earlier noted a series of 2,3-alkanediols in the uropygial glands of birds (Haahti
and Fales, 1967), so 2,3-hexadecanediol diacetate was prepared from the diol syn-
thesized in connection with that study. The 2,3-diol diacetate (Figure 2, bottom
spectrum) did indeed show cleavage between the acetoxy groups producing ions at
m/z 87 and m/z M-87 at 255, but its spectrum was not identical with that of peak 3.
An isomeric ion CH2(OAc)CH2+ of m/z 87 is theoretically available also from 1,3-
hexadecanediol diacetate, so this compound was synthesized by the action of vinyl-
magnesium bromide on tetradecanal followed by hydroboration, oxidation, and
acetylation. This compound (spectrum not shown) showed an abundant -cleavage
peak containing both acetate units at m/z 159 while the intensity at m/z 87 was only
8%, so it was also ruled out. By now it was clear that the two acetate units are more
remotely located in the unknown. Based on the ion at m/z 87, as well as the presence
in the extract of 1,16-hexadecanediol diacetate, we considered a likely candidate to
be the 1,15-diol isomer. This diol was prepared by reacting commercially available
ω-pentadecalactone (Aldrich) with methyllithium to form 1-hexadecanol-15-one

FIG. 2. Mass spectra of peaks 3, 4, and 5 in the gas chromatogram of the abdominal glands
of A. vanillae. 2,3-Hexadecanediol acetate is presented for comparison to other diacetates
(peaks 3 and 4).

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ROSS ET AL.
(with much of the tertiary alcohol 2-methyl-2,15-pentadecanediol as by-product).
Reduction of the ketone with sodium borohydride and acetylation with acetic anhy-
dride gave the desired 1,15-pentadecanediol diacetate, which was indeed identical
in retention time and mass spectrum with the compound represented by peak 3.
Predation on Agraulis vanillae. Predation on adults of A. vanillae, flying in
a butterfly house, was never observed during the nine-month observation period.
More than 400 butterflies were released into the exhibit building that also contained
a population of free-flying insectivorous birds, including two species of aggressive
predators; the superb starling and the red-rumped cacique. In addition, evidence
of detached wings were never found on the ground. However, all species, other
than the those belonging to the subfamily Heliconiinae, were eagerly attacked and
eaten. (In time, many of the predatory birds had to be removed from the exhibit
because they were decimating all nonheliconian butterflies.) Thus, in an enclosed
environment, containing a significant population of predatory birds, adults of A.
vanillae are discriminated against as prey items.
Similar results were obtained in an outdoor butterfly garden in which A.
vanillae was the dominant butterfly species by a factor of at least 10. Different
species of butterflies were observed to be fed upon by invertebrate predators that in-
cluded spiders (Araneidae), assassin bugs (Reduviidae), dragonflies (Libellulidae),
wasps (Vespidae), ambush bugs (Physmatidae), and praying mantids (Mantidae).
Vertebrate predators included anole lizards and a variety of free-flying birds. Preda-
tion on butterfly species, especially Phyciodes tharos (Nymphalidae:Melitaeinae),
Hermeuptychia hermes (Satyridae), and Papilio polyxenes (Papilionidae) was pro-
nounced but was never observed with A. vanillae. In spite of its abundance, A.
vanillae is obviously not a commonly utilized prey species and is avoided by both
invertebrate and vertebrate predators.
DISCUSSION
The only truly volatile component found in both sexes in this study was 6-
methyl-5-hepten-2-one, a well-known ant alarm pheromone and defensive
allomone (Tomalski et al., 1987), and this compound may be objectionable to
birds as well. Use of the volatile ketone as a solitary defensive compound by
both cockroaches and beetles (unpublished data) clearly points to a similar role
in adult A. vanillae. To us, the odor of this ketone, which can be detected easily
when the butterfly is disturbed, does not recall the “phenylcarbamylamine” cited
by Eltringham (1925), but, of course, this is a subjective evaluation.
The simultaneous occurrence of this compound in its alcohol form in the ex-
tract where it is conjugated to palmitic, oleic, and stearic acids may be significant.
It is possible that this constitutes a method for its storage until required, whereupon
it may be available via the sequential action of a lipase and oxidase from another

GLAND CHEMISTRY OF NYMPHALID BUTTERFLY
1227
source or gland. The higher molecular weight diol diacetates may also be involved
here by dissolution of the ketone, reducing its loss through evaporation. An alter-
native possibility for the diol diacetates, for which we have no evidence, is that they
might have a function in the butterfly similar to that of the diols in the preen glands
of fowl, i.e., by rendering their wing coverings water repellent (Haahti and Fales,
1967). However, extensive investigations on the Heliconiinae, implemented more
than 100 years ago (Mu¨ller, 1886), emphasized that the secretions of the abdominal
glands of these nymphalids clearly possessed a defensive function that afforded
the butterflies considerable protection from predators. Although we cannot state
unambiguously that the glandular components of A. vanillae are responsible for
the apparent immunity of these butterflies from avian predation, we suspect that
the compounds in this abdominal exudate may play an important role in defensive
contexts. For example, we have observed that several ant species are effectively
repelled by the defensive secretion of the cockroach Neostylopyga rhombifolia
(unpublished data). This exudate is essentially pure 6-methyl-5-hepten-2-one. In
addition to deterring these predatory invertebrates, we have also observed that the
ketone-rich secretion of N. rhombifolia (Blattidae) effectively repels the American
chameleon, Anolis carolinensis, a very aggressive vertebrate predator. These stud-
ies indicate that 6-methyl-5-hepten-2-one possesses deterrent properties against at
least some invertebrate and vertebrate predators.
Our studies on the level of avian predation on free-flying A. vanillae clearly
demonstrate that this eminently aposematic lepidopteran is virtually immune to
the predatory activities of insectivorous birds. This was particularly striking in a
butterfly conservatory where these lepidopterans shared their flying space with
birds that fed readily on butterfly species other than A. vanillae (the same was true
in the butterfly garden). Observations of A. vanillae versus birds in the butterfly
exhibit indicated that birds took evasive action when they closed on the butterflies.
It appears that these insects possess a powerful organoleptic shield that appears
to insulate them from at least some avian and nonavian predators as well. Since
the odor of 6-methyl-5-hepten-2-one can be detected in the presence of feeding
A. vanillae, it is possible that the secretion of the abdominal glands may normally
leak, thus enabling this aposematic species to advertise its presence freely through
olfaction. Producing this ketone in sufficient quantity in order to be olfactorily
aposematic may be possible if these butterflies are indeed generating its alco-
holic precursor from the rich store of fatty acid esters of 6-methyl-5-hepten-2-ol.
Additionally, it appears as if A. vanillae has morphological strategies that allow
individuals to recover easily from an attack, another indicator that the species is
protected.
The extraordinary tenacity of the individual butterflies that had been subjected
to the milking procedures is probably yet another indication that A. vanillae is
a protected species. Potential predators must learn which species to avoid, and
learning requires sampling. Consequently it is in the best interest of a protected

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prey to be able to survive an occasional attack and so most protected species have
developed morphological strategies that bestow some degree of toughness (Ross,
2000a.).
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