Update on the defensive chemicals of the little black ant, Monomorium minimum (Hymenoptera: Formicidae)

Article abstract of DOI:10.1016/j.toxicon.2016.09.009

Alkaloids, including 2,5-dialkylpyrrolidines and 2,5-dialkylpyrrolines, have been reported to be components in the venom of little black ants, Monomorium minimum (Buckley). Two fatty amines were recently reported as minor compounds. By analyzing the discharge collected from the stinger apparatus (milking), this study revealed the presence of an additional seven compounds in the defensive secretion of this ant species. Compounds identified were 9-decenyl-1-amine, N-methylenedecan-1-amine, N-methylenedodecan-1-amine, 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-pyrroline, N-methyl-2-(hex-5-enyl)-5-nonanyl-1-pyrrolidine, β-springene ((E,E)-7,11,15-trimethyl-3-methylene-1,6,10,14-hexadecatetraene) and neocembrene ((E,E,E)-1-isopropenyl-4,8,12-trimethylcyclotetradeca-3,7,11-triene). β-springene and neocembrene were found only in the defensive secretion of queens. Analyses of the contents of isolated poison and Dufour's glands of the queen indicated that all amines and alkaloids were from the poison gland and that β-springene and neocembrene were from the Dufour's gland. This demonstrated that the defensive secretion in M. minimum queens consists of components from both glands. This is also the first report on the natural occurrence of 9-decenyl-1-amine, N-methylenedecan-1-amine, and N-methyllenedodecan-1-amine.

Full text of DOI:10.1016/j.toxicon.2016.09.009

Toxicon 122 (2016) 127e132
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Update on the defensive chemicals of the little black ant, Monomorium
minimum (Hymenoptera: Formicidae)
,
Jian Chen a *, Charles L. Cantrell b, David Oi c, Michael J. Grodowitz a
a USDA-ARS, National Biological Control Laboratory, 59 Lee Road, Stoneville, MS 38776, USA
b USDA-ARS, Natural Products Utilization Research, University, MS 38677, USA
c USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, 1600 SW 23rd Drive, Gainesville, FL 32608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Alkaloids, including 2,5-dialkylpyrrolidines and 2,5-dialkylpyrrolines, have been reported to be com-
ponents in the venom of little black ants, Monomorium minimum (Buckley). Two fatty amines were
recently reported as minor compounds. By analyzing the discharge collected from the stinger apparatus
(milking), this study revealed the presence of an additional seven compounds in the defensive secretion
of this ant species. Compounds identified were 9-decenyl-1-amine, N-methylenedecan-1-amine, N-
methylenedodecan-1-amine, 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-pyrroline, N-methyl-2-(hex-5-enyl)-
Received 13 June 2016
Received in revised form
13 September 2016
Accepted 14 September 2016
Available online 15 September 2016
5-nonanyl-1-pyrrolidine,
and neocembrene ((E,E,E)-1-isopropenyl-4,8,12-trimethylcyclotetradeca-3,7,11-triene).
neocembrene were found only in the defensive secretion of queens. Analyses of the contents of isolated
poison and Dufour's glands of the queen indicated that all amines and alkaloids were from the poison
gland and that b-springene and neocembrene were from the Dufour's gland. This demonstrated that the
b
-springene ((E,E)-7,11,15-trimethyl-3-methylene-1,6,10,14-hexadecatetraene)
-springene and
Keywords:
Little black ants
Venom
Poison gland
Dufour's gland
b
defensive secretion in M. minimum queens consists of components from both glands. This is also the first
report on the natural occurrence of 9-decenyl-1-amine, N-methylenedecan-1-amine, and N-methyl-
lenedodecan-1-amine.
Published by Elsevier Ltd.
1. Introduction
gland secretion may be accompanied by chemicals from Dufour's
gland. The venom is used by M. minimum workers in competition
The red imported fire ant, Solenopsis invicta Buren, is an invasive
ant species that was introduced into the United States from South
America in the 1930's and has become ecologically dominant in
areas it infests. Several native ant species have been reported to
compete effectively with S. invicta (Baroni Urbani and Kannowski,
1974; Helms and Vinson, 2001; Rao and Vinson, 2004); among
them is the little black ant, Monomorium minimum (Buckley)
(Smith, 1965; Thompson, 1990). Little black ants can build their
nests close to fire ant colonies and successfully compete with S.
invicta for food and territory (Thompson, 1990; Rao and Vinson,
2004). Like S. invicta, both workers and queens of M. minimum
possess a venom apparatus, also known as a stinger apparatus. The
venom apparatus is composed of the sting and two glands, the
venom gland and Dufour's gland. When discharged, the venom
with other ants for food and territory (Adams and Traniello, 1981).
M. minimum venom has been shown to repel S. invicta workers
(Baroni Urbani and Kannowski, 1974) and may be one of several
important factor why M. minimum can compete effectively against
S. invicta.
Alkaloids are the dominant components of M. minimum venom,
including; saturated and unsaturated 2,5-dialkyl-pyrrolidines
(Jones et al., 1982; Lange et al., 1989), unsaturated 2,5-dialkyl-pyr-
rolines (Lange et al., 1989), and unsaturated N-methyl-2,5-dialkyl-
pyrrolidine (Jones et al., 1982). Two primary amines, decylamine
and dodecylamine were recently reported (Wang and Chen, 2015)
(see Table 1 for the details of compounds that have been published
for M. minimum).
It is not unusual for venom compositions to differ between
workers and queens, particularly in the proportion of some major
components (Maile et al., 2000; Eliyahu et al., 2011; Chen et al.,
2012). However, research on venom chemistry in Monomorium
species have mainly focused on workers. As part of our effort in
searching for insecticidal substances from natural products, we
* Corresponding author. USDA-ARS, BCPRU, 59 Lee Road, Stoneville, MS 38776,
USA.
E-mail address: jian.chen@ars.usda.gov (J. Chen).
http://dx.doi.org/10.1016/j.toxicon.2016.09.009
0041-0101/Published by Elsevier Ltd.

128
J. Chen et al. / Toxicon 122 (2016) 127e132
Table 1
Compounds that have been previously identified in the venom of little black fire ants, Monomorium minimum.
Compounds
R1
R2
References
(CH2)4CH]CH2
(CH2)4CH]CH2
(CH2)7CH]CH2
(CH2)8CH3
Lange et al., 1989
(CH2)4CH]CH2
(CH2)4CH]CH2
(CH2)5CH3
(CH2)7CH]CH2
(CH2)8CH3
(CH2)8CH3
Jones et al., 1982; Lange et al., 1989
Lange et al., 1989
Lange et al., 1989
(CH2)4CH]CH2
(CH2)7CH]CH2
Jones et al., 1982
Wang and Chen, 2015
Wang and Chen, 2015
analyzed chemical components in the venom of M. minimum for
both workers and queens. We report here the discovery of previ-
ously unknown compounds and differences in venom composition
between workers and queens.
the GC-MS.
2.3. Gland content analysis
The content of poison and Dufour's glands were analyzed
separately for three queens from each of two colonies. For gland
removal, queens were pinned dorsal side up on a raised wax-based
dissecting tray using a stereo microscope at 7 to 10ꢁ magnification.
Individuals were secured through the pronotum using a minuten
pin and the abdomen immobilized by laying pins cross-ways over
the narrow pedicle. The poison and Dufour's gland were removed
by grasping the terminal abdominal segments or the stinger itself
with a fine forceps and pulling posteriorly. After removal, the entire
venom apparatus was placed in a drop of phosphate buffered saline
on a microscope slide. The poison and Dufour's glands were sepa-
rated using Vannas Spring Scissors with a 2 mm cutting edge. After
2. Materials and methods
2.1. Ants
Ten colonies of Monomorium minimum were collected from
Washington County, Mississippi. Colonies were reared in plastic
trays with distilled water, 10% w/w sugar water, and mealworm
beetle pupae, Tenebrio molitor, under laboratory conditions at 28 ꢀC
and 45% RH.
2.2. Venom composition
separation each gland was placed in 100 ml dichlormethane and a
two microliter sample injected into the GC-MS system. While the
poison/Dufour's gland apparatus was successfully removed from
worker ants a clear delineation of the two was not achieved due to
the extremely small size of the workers.
Venom is defined here as the discharge released by ants through
the stinger during manual stimulation. Venom was collected
directly on a fiber used for solid phase microextraction (SPME)
(fused silica fiber with 85 m
m polyacrylate, Supelco®, Bellefonte,
PA). A fine forceps was used to hold an ant until a drop of venom
formed on the tip of its stinger (Fig. S1). Under a stereo microscope,
the venom was collected by touching the drop with a SPME fiber.
The ant was then stimulated to release more venom by gently
touching the stinger using the fiber. At least three drops were
collected on a fiber from each individual ant. The sample was then
directly analyzed using gas chromatographyemass spectrometry
(GC-MS). The SPME fiber was injected into the GC injection port
within 30 s of venom collection. The absolute concentration of each
compound was not determined; however, relative abundances
were estimated based on the GC TIC (total ion chromatogram) peak
areas without using correction factors. For SPME samples, some
alkaloid peaks were not well separated due to their higher con-
centrations. In order to obtain relative peak area of each alkaloid in
relation to the total alkaloid, diluted hexane ant extraction was
used to ensure adequate separation of each alkaloid peak in the
total ion chromatogram. For those samples, one ant was placed in a
2.4. Gas chromatography e mass spectrometry
The GC-MS system consisted of an Agilent 7890A gas chro-
matograph with a HP-5 capillary column (30 m ꢁ 0.25 mm i.d.,
0.25 mm film thickness) and an Agilent 5975 mass selective de-
tector. The GC temperature was programmed at an initial temper-
ature of 50 ꢀC, held for 1 min, increased to 240 ꢀC at a rate of 20 ꢀC/
min and held for 20 min, and then increased to 280 ꢀC and held for
8 min. Splitless mode was used. Injection temperature was 250 ꢀC,
and transfer line temperature was 270 ꢀC. The mass spectrometer
was operated at 70 eV in the electron impact mode. Chemical
identification was confirmed by comparing the retention times and
mass spectra of samples with those of standards that were either
purchased commercially or synthesized in our laboratory. 9-
decenyl-1-amine was synthesized and
b
-springene (purity ꢂ 95%)
was purchased from Allichem LLC (Baltimore, Maryland, USA). If
standards were not available, identification was made by compar-
ison of their mass spectra with those in either NIST library (National
Institute of Standards and Technology, USA) or the literature.
200
Inc., Vineland, New Jersey, USA) with 100
then crushed using a micropipette and 2 l liquid was injected into
ml glass conical limited volume insert (J.G Finneran Associates,
ml hexane. The ant was
m

J. Chen et al. / Toxicon 122 (2016) 127e132
129
2.5. Synthesis of 9-decenyl-1-amine
hydrogen gas evolution ceased. After further addition of 10 mL H2O
and 5 mL of 20% aq. NaOH solution, the suspension filtered over a
Büchner funnel. The residue was rinsed with 25 mL THF. The fil-
trates were combined and the solvents were evaporated in a vac-
uum to a volume of about 30 mL. 50 mL of CH2Cl2 and 50 mL of H2O
were added, the organic phase separated and dried over MgSO4.
The solvents were evaporated in a vacuum to yield compound 4
(0.406 g, 2.61 mmol, 58%) as a light yellow oil, which solidified over
The synthesis scheme for 9-decenyl-1-amine is illustrated in
Fig. 1. For synthesis of 1-bromo-9-decene (2), 9-decen-1-ol (1,
0.506 g 3.24 mmol, Sigma-Aldrich, St. Louis, MO) was dissolved in
10 mL of CH2Cl2 in a 50 mL three-necked round bottom flask
equipped with a condenser. After addition of tetrabromoethane
(1.2806 g, 3.86 mmol), the reaction mixture was cooled to 0 ꢀC and
triphenylphosphine (1.0124 g, 3.86 mmol) was added over a period
of 30 min. The mixture was warmed to room temperature and
refluxed overnight. The reaction mixture was cooled to room
temperature and 10 mL of pentane was added. Triphenylphosphine
oxide precipitated as a colorless solid. The suspension was filtered
over a 30 mL fritted funnel and the residue was washed with
pentane (3 ꢁ 10 mL). The filtrate was evaporated under vacuum to
yield a colorless oil. The residue was separated by silica gel column
3 days (1H NMR (500 MHz in CDCl3),
(1H, J ¼ 17), 4.91 d (1H, J ¼ 10), 2.77 t (1H, J ¼ 7), 2.00e2.04 (m, 2H),
1.53e1.57 (m), 1.27 (s) 13C NMR (125 MHz, CDCl3)
139.04, 114.16,
d 5.75e5.83 (m, 1H), 4.97 d
d
77.33, 77.08, 76.82, 41.11, 33.76, 31.06, 29.41, 29.29, 29.05, 28.88,
26.80. EI-MS (70eV): 55 (9), 56 (8), 114 (5), 67 (5), 86 (4)).
3. Results
chromatography on a Biotage 100þM column (40e63
mm, 60 Å,
3.1. Venom of workers
39 ꢁ 157 mm) running at 50 mL/min using a hexane: EtOAc
gradient beginning with 100:0 to 80:20 over 1540 mL, followed by
80:20 to 0:100 over 1540 mL. 22 mL fractions were collected and
recombined based on TLC similarities into compound 2 (0.650 g,
In addition to 2,5-dialkylpyrrolines, 2,5-dialkylpyrrolines and
fatty amines, 9-decenyl-1-amine, N-methylenedecan-1-amine and
N-methylenedodecan-1-amine were identified (Figs. 2 and 3). The
identity of 9-decenyl-1-amine was confirmed using the synthetic
standard. Both retention time and mass spectra were matched
between the sample and standard (Fig. S2). The identity of N-
methylenedecan-1-amine and N-methylenedodecan-1-amine
were confirmed by direct comparison of the MS with the corre-
sponding compound in the NIST library search (Figs. S3 and S4).
Three 2,5-dialkylpyrrolines, three 2,5-dialkylpyrrolidines, and two
2.97 mmol, 92%). (1H NMR (400 MHz in CDCl3),
d 5.74e5.84 (m, 1H),
4.96 dd (2H, J ¼ 2, 17), 4.92 dd (2H, J ¼ 2, 10), 3.38 t (2H, J ¼ 7),
1.99e2.05 (m, 2H), 1.79e1.87 (m), 1.28 (s) 13C NMR (100 MHz,
CDCl3)
d 139.32, 114.39, 77.55, 77.23, 76.91, 34.22, 33.98, 33.04,
29.49, 29.21, 29.08, 28.93, 28.37. EI-MS (70eV): 55 (17), 69 (11), 97
(11), 83 (6), 148 (4)).
For synthesis of 1-azidodec-9-ene (3), 1-bromo-9-decene (2,
0.625 g, 2.85 mmol) was dissolved in 5 mL dry DMSO in a 50 mL
Schlenk flask under inert gas atmosphere and sodium azide
(0.368 g, 5.67 mmol) was added. The reaction mixture was stirred
at room temperature for 30 min and then heated to 60 ꢀC for 2 h.
The mixture was cooled to room temperature and 5 mL of CH2Cl2
and 5 mL of H2O were added. The organic phase was separated,
washed with water (3 ꢁ 5 mL) and dried with Na2SO4. The solvents
were evaporated in a vacuum to yield compound 3 as a light yellow
oil, which was used in the following reaction step without further
purification (0.326 g, 1.79 mmol, 63%). (1H NMR (400 MHz in
CDCl3),
J ¼ 2,10), 3.21 t (2H, J ¼ 7), 1.97e2.03 (m, 2H), 1.52e1.59 (m),1.26 (s)
13C NMR (100 MHz, CDCl
139.24, 114.32, 77.55, 77.23, 76.91,
d
5.72e5.82 (m, 1H), 4.95 dd (2H, J ¼ 2, 17), 4.91 dd (2H,
)
d
3
51.61, 33.91, 29.45, 29.24, 29.14, 29.01, 28.97, 26.84. EI-MS (70eV):
55 (11), 122 (8), 70 (8), 69 (6), 56 (6)).
For synthesis of 9-decen-1-amine (4), in a 250 mL three necked
round bottom flask equipped with a condenser and 50 mL of dry
THF were cooled to 0 ꢀC in an ice/water bath. LiAlH4 (0.303 g,
7.98 mmol) was added slowly to produce a grey suspension, which
was stirred for 5 min 1-azido-9-decene (3, 0.818 g, 4.51 mmol) was
dissolved in 10 mL dry THF and slowly added to the LiAlH4 sus-
pension over a period of 20 min, stirred for another 30 min at 0 ꢀC
and then refluxed for an additional 1.5 h. The reaction mixture was
cooled to 0 ꢀC and water was subsequently added drop-wise until
Fig. 2. New amines and terpenes identified in the venom of little black ants Mono-
morium minimum. (E,E)-7,11,15-trimethyl-3-methylene-1,6,10,14-hexadecatetraene (b-
springene) and (E,E,E)-1-isopropenyl-4,8,12-trimethylcyclotetradeca-3,7,11-triene
(neocembrene) were found only in queens.
Fig. 1. Synthesis scheme for 9-decenyl-1-amine (4).

130
J. Chen et al. / Toxicon 122 (2016) 127e132
nonanylpyrrolidine (Fig. S12). The relative peak areas of these
identified compounds are shown in Table S1 and Fig. S15. The
venom composition seemed to be dominated by 2-(hex-5-enyl)-5-
(1-non-8-enyl)pyrrolidine
(39.0%)
and
2-(hex-5-enyl)-5-
nonanylpyrrolidine (55.4%). Since the relative peak area of 2-(1-
non-8-enyl)-5-(1-hex-5-enyl)-1-pyrroline and N-methyl-2-(hex-
5-enyl)-5-nonanyl-1-pyrrolidine was very low (<0.004%) and not
detected in every sample, they were not included in calculating the
relative peak area of each alkaloid (Fig. 4). All amines and alkaloids
found in the whole worker extract also exist in the collected venom.
3.2. Venom of queens
The
queen
venom
did
not
contain
2-hexyl-5-
nonanylpyrrolidine. In addition to 2,5-dialkylpyrrolines, 2,5-
dialkylpyrrolines, decylamine, and dodecylamine; 9-decenyl-1-
amine, N-methylenedecan-1-amine and N-methylenedodecan-1-
amine, b-springene ((E,E)-7,11,15-trimethyl-3-methylene-1,6,10,14-
hexadecatetraene) and neocembrene ((E,E,E)-1-isopropenyl-
4,8,12-trimethylcyclotetradeca-3,7,11-triene) were also identified.
Identity of
b-springene was confirmed by a synthetic standard
(Fig. S13). The identity of neocembrene was confirmed by
comparing the mass spectrum of the sample with that in the
literature. This compound has been reported in the queen of the
pharaoh ant, Monomorium pharaonis (Edwards and Chambers,
1984). Extracts of M. pharaonis were also analyzed using the same
GC-MS conditions. The retention time and mass spectrum of neo-
cembrene in M. pharaonis were matched to those of neocembrene
in M. minimum queen (Fig. S14). The relative peak areas of identi-
fied compounds are shown in Table S1. The venom composition
seemed to be dominated by 2-(hex-5-enyl)-5-(1-non-8-enyl)pyr-
rolidine (81.3%), followed by 2-(hex-5-enyl)-5-nonanylpyrrolidine
(7.5%) (Fig. S15). Since 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-
pyrroline and N-methyl-2-(hex-5-enyl)-5-nonanyl-1-pyrrolidine
was detected in only a minimal number of queen venom samples,
they were not included in calculating the relative peak area of each
Fig. 3. Alkaloids found in the venom of the little black ants, Monomorium minimum. A,
B, C, D, and E were detected in every sample of both workers and queens and F only in
workers. G and H was observed in both workers and queens, but not in every sample.
Relative peak area of each alkaloid in the peak area of total alkaloids is shown in
Fig. S15.
alkaloid (Fig. S15). b-springene and neocembrene were only found
in the Dufour's gland. Stinger apparatus with poison and Dufour's
glands of M. minimum queen is shown in Fig. 4.
4. Discussion
To our knowledge, 9-decenyl-1-amine, N-methylenedecan-1-
amine, and N-methyllenedodecan-1-amine have never been re-
ported as naturally occurring compounds. Two fatty amines,
decylamine and dodecylamine, from M. minimum were reported to
be insecticidal against the red imported fire ants (Wang and Chen,
2015). Dodecylamine repelled the common house fly, Musca
domestica and decylamine repelled the honey bee, Apis mellifera
(Atkins et al., 1975). Dodecylamine deterred oviposition of Euro-
pean corn borer, Ostrinia nubilalis (Binder and Robbins, 1997).
Decylamine and dodecylamine also showed high nematicidal ac-
tivity against pine wood nematode, Bursaphelenchus Lignicolus
(Nagase et al.,1982). Due to its similarity in structure to decylamine,
9-decenyl-1-amine may also have similar toxic and repellant ef-
Fig. 4. Poison and Dufour's glands in Monomorium minimum queen. b-springene and
neocembrene were only found in the Dufour's gland.
fects.
Both
N-methylenedecan-1-amine
and
N-methyl-
lenedodecan-1-amine have a carbon-nitrogen double bond in their
chemical structures, an imine functional group. Toxicity data of
these two amines are not available in the literature. What functions
they may serve in the ant venom warrants further research.
This study has demonstrated a clear difference in venom
N- 2,5-dialkylpyrrolidines were identified, including 2-(1-non-8-
enyl)-5-(1-hex-5-enyl)-1-pyrroline (Fig. S5), 2-(1-hex-5-enyl)-5-
(1-non-8-enyl)-1-pyrroline (Fig. S6), 2-(1-hex-5-enyl)-5-nonanyl-
1-pyrroline (Fig. S7), 2-(hex-5-enyl)-5-(1-non-8-enyl)pyrrolidine
(Fig. S8), 2-(hex-5-enyl)-5-nonanylpyrrolidine (Fig. S9), 2-hexyl-5-
nonanylpyrrolidine (Fig. S10), N-methyl-2-(hex-5-enyl)-5-(1-non-
8-enyl)pyrrolidine (Fig. S11), and N-methyl-2-(hex-5-enyl)-5-
composition between workers and queens.
cembrene were found only in the venom of queens while 2-hexyl-
5-nonanylpyrrolidine did not occur in the queen. -springene, a
diterpene, was originally isolated from a skin (dorsal) gland of the
b-springene and neo-
b

J. Chen et al. / Toxicon 122 (2016) 127e132
131
springbok antelope, Antidorcas marsupialis, from Africa (Burger
et al., 1978, 1981). It has been found in other mammals such as
the white-lipped peccary, Tayassu pecari (Waterhouse et al., 2001),
and collared peccary, Tayassu tajacu (Waterhouse et al., 1996); two
reptilians, Cuvier's dwarf caiman, Paleosuchus palpebrosus, and the
smooth-fronted caiman, Paleosuchus trigonatus (Schulz et al.,
2003); four species of oribatid mites, Oribotritia banksi, O. berlesei,
O. hermanni, and O. storkani (Raspotnig et al., 2011); five species of
bumblebees, Bombus griseocollis (Bertsch et al., 2004), B. hypnorum
(Cahlikova et al., 2004), B. morrisoni, B. rufocinctus (Bertsch et al.,
2008), B. semenoviellus (Hovorka et al., 2006); and two stingless
enyl)pyrrolidine were reported in M. minimum by Jones et al.
(1982). Lange et al. (1989) reported the following 5 alkaloids in
this species, including 2-(1-hex-5-enyl)-5-(1-non-8-enyl)-1-
pyrroline, 2-(1-hex-5-enyl)-5-nonanyl-1-pyrroline, 2-(hex-5-
enyl)-5-(1-non-8-enyl)pyrrolidine,
2-(hex-5-enyl)-5-
nonanylpyrrolidine, and 2-hexyl-5-nonanylpyrrolidine. The pres-
ence of all these alkaloids were confirmed in this study. In addition,
it is the first time that 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-
pyrroline and N-methyl-2-(hex-5-enyl)-5-nonanyl-1-pyrrolidine
were identified in the venom of M. minimum. However, both al-
kaloids have been found in other ant species in the genus of Mon-
omorium. 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-pyrroline was
found in M. near metoecus, M. virdum, and M. ebeninum and N-
methyl-2-(hex-5-enyl)-5-nonanyl-1-pyrrolidine in M. near metoe-
cus, M. virdum, M. near emersoni, and M. cyaneum (Jones et al.,
1982). Both 2,5-dialkyl-pyrrolidines and 2,5-dialkyl-pyrrolines are
insecticidal (Bacos et al., 1988).
This study demonstrates that the venom in M. minimum queen
consists of products from both the poison glands and the Dufour's
glands. Due to the extremely small size of the workers, separation
of the poison gland from the Dufour's gland in workers was not
successful. Except 2-hexyl-5-nonanylpyrrolidine, all compounds
found in worker's venom were also found in queen's poison gland.
Although the possibility of Dufour's gland origin couldn't be
completely ruled out, all the identified compounds for workers are
most likely from the poison gland.
ꢀ
bees, Melipona beecheii (Cruz-Lopez et al., 2005) and Nannotrigona
testaceicornis (Pianaro et al., 2009). b-springene was also found in
the Dufour's gland of the ectoparasitoid Habrobracon hebetor (Say)
where its abundance varied significantly with age (Howard et al.,
2003). The only ant species that has been reported to produce
springene is the Old World army ant, Aenictus rotundatus (Oldham
et al., 1994). However, its isomer, E,E,E- -springene, was found in
b-
a
the Dufour's gland of the Dinosaur ant, Nothomyrmecia macrops
(Billen et al., 1988). The secretion of Dufour's gland of ants can have
multiple functions (Morgan, 2008). The potential function of
b-
springene in the venom of M. minimum queens can only be eluci-
dated by further experimentation.
There are several synonyms for neocembrene. The chemical
structure of neocembrene was first assigned by Shmidt et al. (1970)
while studying the diterpene hydrocarbons of the oleoresin in Si-
berian spruce, Picea obvata (Ledb.) and Korean pine, Pinus koraensis
(Sieb Zucc.). Neocembrene was found by Moore (1966) as the trail
pheromone of Nasutitermes exitiosus (Hill); however, the chemical
structure of neocembrene in N. exitiosus was not elucidated until
1972 (Birch et al., 1972). Due to a slight difference in NMR spectrum
compared to those of neocembrene from two conifers mentioned
above, it was then named as neocembrene-A. Neocembrene was
also isolated from the gum-resin of Commiphora mukul (Hook, ex
Stocks) Engl and it was named as cembrene-A (Patil et al., 1973).
Since its discovery, it has been found in numerous plants, corals,
insects, and mammals. Vanderah et al. (1978) found it in soft coral,
Nephthea species. In addition to N. exitiosus, neocembrene was
found in 16 other termite species which are belong to 7 different
genera, including Constrictotermes cyphergaster (Silvestri), Nasuti-
termes corniger (Motschulsky), N. ephratae (Holmgren), N. exitiosus,
N. guayanae, N. kemneri, N. lujae, N. voeltzkowi, Trinervitermes
Defense is very important to the survival of an ant colony. Many
ant species depend on chemicals for their defense. The results of
this study extend our knowledge about chemicals produced in the
venom of M. minimum, which will help advance our understanding
of its biology.
Ethical Statement
Not applicable.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
ꢁ
geminates (Sillam-Dusses et al., 2010), and T. trinervoides. (Sillam-
We thank Dr. Hamed K. Abbas, Biological Control of Pests
Research Unit, USDA-ARS, Stoneville, MS and Dr. Aijun Zhang,
Invasive Insect Biocontrol and Behavior Laboratory, USDA-ARS,
Beltsville, MD for critical review of this manuscript. We thank Mr.
Leon Hicks for his technical assistance. Mention of trade names or
commercial products in this publication is solely for the purpose of
providing specific information and does not imply recommenda-
tion or endorsement by the U. S. Department of Agriculture.
ꢁ
Dusses et al., 2010), Cubitermes umbratus (Wiemer et al., 1979),
Crenetermes mixtus (Prestwich, 1979), N. costalis (Hall and Traniello,
1985), Noditermes wasambaricus (Meinwald et al., 1978), Pro-
rhinotermes canalifrons (Sillam-Dusses et al., 2005), P. simplex
(Sillam-Dusses et al., 2009), T. bettonianus (McDowell and Oloo,
ꢁ
ꢁ
1984). Neocembrene was found in only two insect species outside
isoptera: a sandfly, Lutzomyia longipalpis (Hamilton et al., 2004) and
a myrmicine ant, Monomorium pharaonis (Edwards and Chambers,
1984). Neocembrene has exhibited cytotoxicity against A549 (hu-
man lung adenocarcinoma), HT-29 (human colon adenocarci-
noma), KB (human epidermoid carcinoma), and P-388 (mouse
lymphocytic leukemia) cell lines (Duh et al., 1999). In M. pharaonis,
neocembrene was found only in the Dufour's gland of the fertile
queens. Since alates and young queens do not contain neo-
cembrene, its potential role in suppressing the development of new
sexual forms (males and queens) in an ant colony was suggested
(Edwards and Chambers, 1984). The function of neocembrene in M.
minimum is another interesting area and more research is
warranted.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.toxicon.2016.09.009.
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