Influence of Mating Type and Oviposition Period on Mandibular ...

<strong>Influence</strong> <strong>of</strong> <strong>Mating</strong> <strong>Type</strong> <strong>and</strong> <strong>Oviposition</strong> <strong>Period</strong> on M<strong>and</strong>ibular
Pheromone Levels in Apis melli/era L. Honeybee Queens
Abdulaziz S. AI-Qarni"", P. Larry Phelan, Brian H. Smith, <strong>and</strong> Susan W. Cobey
Dept. <strong>of</strong> Entomology, Ohio State Ulliversity, Columbus, Ohio 43210, USA
""CurrentAddress: Dept. <strong>of</strong> Plant Protectioll, College <strong>of</strong> Food Science <strong>and</strong> Agriculture,
King Saud Universihj, Riyadh, 11451, P.O. Box - 2460, Saudi Arabia
Abstract. M<strong>and</strong>ibular gl<strong>and</strong>s <strong>of</strong> Naturally Mated (NM)<strong>and</strong> Instrumentally Inseminated (II)Apis mellifera L.
queens were extracted after 1 <strong>and</strong> 2-weeks <strong>of</strong> oviposition <strong>and</strong> bioassayed against worker honeybees. NM
extracts evoked higher response than II extracts with no effect for week or interaction between week <strong>and</strong>
mating type. M<strong>and</strong>ibular gl<strong>and</strong>s analysis <strong>of</strong> NM II honeybee queens revealed presence <strong>of</strong> all QMP
components except HVA. Significantlyhigher amount <strong>of</strong> the QMP major component 9-keto-(E)-2-decenoic
acid (9-0DA) was recorded in NM queens than in II queens whereas oviposition period did not present
significant changes in QMP levels at 1 <strong>and</strong> 2 weeks <strong>of</strong> oviposition. Significant differences were observed in
proportions <strong>of</strong> 9-0DA <strong>and</strong> ±9-HDA<strong>of</strong> NM queens after one week <strong>of</strong> oviposition.
Key word: Honeybee Queen, Natural <strong>Mating</strong>, Instrumental Insemination, Queen M<strong>and</strong>idular Pheromone,
Bioassay,<strong>Oviposition</strong>, Chemicalanalysis, Apis mellifera L.
Semichemicals which are produced by one
organism incite a response in other organisms
are the main channel for communication in
honeybees. As eusocial insects, honeybees
represent the most advanced level <strong>of</strong> sociality
among invertebrates. In this system, the queen
influences the physiology <strong>and</strong> behavior <strong>of</strong> the
colony workers by the release <strong>of</strong> several volatile
<strong>and</strong> non-volatile chemicals from a number <strong>of</strong>
exocrine gl<strong>and</strong>s. This eventually brings the
whole colony to a natural state <strong>of</strong> cohesion,
reproduction <strong>and</strong> productivity.
Sources <strong>of</strong> queen pheromones include
m<strong>and</strong>ibular, tergal, Koshevnikov, Dufour, <strong>and</strong>
tarsal gl<strong>and</strong>s. Some <strong>of</strong> these gl<strong>and</strong>ular
semiochemicals are yet to be behaviorally <strong>and</strong>
chemically characterized. M<strong>and</strong>ibular <strong>and</strong>
tergal gl<strong>and</strong> secretions are the most studied
substances. Tergal gl<strong>and</strong> secretions <strong>of</strong> two
African honeybee races (Apis mellifera capensis
<strong>and</strong> Apis mellifera scutellata) were chemically
identified (Wossler <strong>and</strong> Crewe, 1999a).
Behavioral bioassays were conducted on the
whole tergal extracts (Wossler <strong>and</strong> Crewe,
1999b), but not on identified chemicals.
The only gl<strong>and</strong> secretions that have been
behaviorally bioassayed, <strong>and</strong> chemically
identified <strong>and</strong> synthesized are the queen
m<strong>and</strong>ibular secretions (Siessor et al., 1988). Five
components are found to elicit primer <strong>and</strong>
releaser effects on other colony members; the
queen m<strong>and</strong>ibular pheromone (QMP) is
comprised <strong>of</strong> three aliphatic acids <strong>and</strong> two
aromatic compounds: 9-keto-(E)2-decenoic acid
(9-0DA), (R,E)-(-) <strong>and</strong> (S,E)-(+)-9-hydroxy-2-
decenoic acid (9-HDA), methyl p-hydroxybenzoate
(HOB), <strong>and</strong> 4-hydroxy-3-methoxyphenylethanol
(HVA) (Siessor et ai., 1988; Plettner et
al., 1996; Engels et ai., 1997). The amount <strong>and</strong>
proportion <strong>of</strong> each individual component
produced by a queen differs by race,
reproductive status <strong>and</strong> age. European mated
queens have significantly higher proportions <strong>of</strong>

9-0DA, HOB, <strong>and</strong> HVA in the QMS bouquet,
while Africanized mated queens have
significantly higher proportions <strong>of</strong> (+) <strong>and</strong> (-) 9-
HDA. Virgin European queens have a higher
proportions <strong>of</strong> 9-0DA, while mated European
queens have roughly equal proportions <strong>of</strong> 9-
ODA <strong>and</strong> 9-HDA. The average amount <strong>of</strong> the
QMP found in a pair <strong>of</strong> m<strong>and</strong>ibular gl<strong>and</strong>s <strong>of</strong> a
European mated queen was found to be: 200 I.l
9-0DA, 80 I.lg9-HAD, 20 I.lgHOB, <strong>and</strong> 2 I.lg
HVA (Pankiw et al., 1996).
Functions <strong>of</strong> queen m<strong>and</strong>ibular pheromones
include both primer <strong>and</strong> releaser effects
on worker bees. As a primer pheromone, it was
found to inhibit queen rearing by workers
(Butler, 1954, 1960; Butler <strong>and</strong> Simpson, 1958;
Winston et al., 1989, 1990; Pettis et al., 1995),
stimulate pollen foraging <strong>and</strong> brood rearing in
small, newly founded colonies (Higo et al.,
1992), inhibit worker juvenile hormone OH)
biosynthesis in laboratory experiments (Kaatz
et al., 1992), <strong>and</strong> regulate the transition <strong>of</strong>
worker activity from within-hive to foraging
duties by lowering their juvenile hormone titers
in colonies (Pankiwe et al., 1998). Contrary to
earlier findings, Willis et al., (1990) reported
that QMP does not affect ovarian development
in worker bees. The releaser effects <strong>of</strong> QMP
include attraction <strong>of</strong> workers to form retinue
around the queen inside the colony (Velthuis,
1967; Winston et al., 1982, 1989; Slessor et al.,
1988; Kaminski et al., 1990), attraction <strong>of</strong>
workers during swarming outside the colony
(Velthuis <strong>and</strong> Van Es, 1964; Butler <strong>and</strong>
Simpson, 1967; Winston et al., 1989), <strong>and</strong>
attraction <strong>of</strong> drones to the queen during mating
flights (Butler <strong>and</strong> Fairey, 1964; Loper et al.,
1996).
As the main channel <strong>of</strong> communication,
queen pheromones have critical importance
during introduction <strong>of</strong> a new mated queen to a
queenless colony. Beekeepers are advised to
replace their queens annually to maintain goodquality
performance for their queens <strong>and</strong>
colonies <strong>and</strong> to maintain low levels <strong>of</strong>
Africanization in commercial colonies.
Superseded queens that have mated with
Africanized drones can result in colonies with
non-commercial properties (Guzman-Nova et
al., 1998) such as high defensive proclivity, high
rate <strong>of</strong> swarming <strong>and</strong> probability to abscond
(Collins et al., 1982; Otis, 1982; Winston et al.,
1979).
Colonies that are led by young queens « 1
yr. old) produce more honey than colonies led
by old queens (Kostarelou-Demianidou et al.,
1995). Old queens' progenies could also have
undesirable characteristics (e.g. high succeptibility
to diseases <strong>and</strong> strong defensive
behavior), which prompts beekeerpers to
replace them with younger ones (Free, 1987).
This regularly performed procedure requires a
period <strong>of</strong> queenlessness on workers bees prior
to introducing a new queen to the colony.
However, worker bees in a queenless colony
become more excited <strong>and</strong> sensitive to
examinations as the queenlessness period
continues. They aggressively attack a foreign
introduced queen by the act <strong>of</strong> mounting,
biting, pulling, <strong>and</strong> stinging. Eventually, this
initiates the balling behavior when workers
start to form a cluster <strong>of</strong> bees around the queen,
usually resulting in queen elimination (Yavada,
1970;Boch <strong>and</strong> Morse, 1974;Pettis et al., 1998).
In spite <strong>of</strong> reducing workers' rejection by
caging foreign queens during introduction for
24 hr, this natural behavior <strong>of</strong> workers was
found to be more frequent towards
instrumentally inseminated queens (II) than
naturally mated queens (NM). II queens have
been reported to have problems with initial
introduction <strong>and</strong> acceptance, <strong>and</strong> with early
supersedure (Smith et al., 1993; Harbo <strong>and</strong>
Szabo, 1984). II queens were also found to have
lower oviposition rates than NM queens
(Harbo, 1986a). Nevertheless, the use <strong>of</strong>
instrumental insemination for honeybee queens
is <strong>of</strong> great value to the beekeeping industry as a
tool for bee breeders <strong>and</strong> queen producers to
increase the quality <strong>of</strong> honeybee races. Natural

mating can result in bees with undesirable
characters that might come from drones <strong>of</strong>
unknown origin.
The study was carried during Spring, 2000
to ascertain the effect <strong>of</strong> mating type <strong>and</strong>
oviposition period <strong>of</strong> queens on the levels <strong>of</strong>
m<strong>and</strong>ibular pheromone. It was hypothesized
that NM queens have higher amounts <strong>of</strong>
m<strong>and</strong>ibular secretion or a different proportion
<strong>of</strong> components than II queens, a difference that
might contribute to the reported lower
acceptance <strong>and</strong> survival rates <strong>of</strong> II queens in
field colonies.
European Apis mellifera carnica queens were
reared by commercial queen rearing method
(Doolittle, 1989) from a Carniolan Gene Pool
(New World Carniolan) maintained by he
Rothenbuhler Honeybee Research Laboratory
at the Ohio State University, Columbus, Ohio.
A group <strong>of</strong> identical mature queen cells were
treated as follows: one half <strong>of</strong> the mature queen
cells ready for emergence were introduced to 5-
frame nucleus colonies containing young
queenless workers with sealed brood, honey
<strong>and</strong> pollen. After emergence, virgin queens
were allowed to mate naturally. The other half
<strong>of</strong> the mature queen cells were kept in an
incubator at 34°C <strong>and</strong> 75-80% RH until
emergence. After 5-days <strong>of</strong> storage in queen
banks, they were instrumentally inseminated
with 8 III<strong>of</strong> semen per queen (Harbo, 1968b) on
the same day on which NM queens are
expected to mate. After inseminatioin, II queens
were caged individually <strong>and</strong> left in queen
banks for 24 hr. The following day, they were
narcotized using C02 so they would lay eggs
earlier (Mackensen, 1947), then they were
introduced to nucleus colonies similar to those
described earlier <strong>and</strong> were released after 48 hr.
Both NM <strong>and</strong> II queens observed 7-10 days for
oviposition. Once eggs were found, 7queens
from each group were collected individually
after one <strong>and</strong> two weeks <strong>of</strong> oviposition for
dissection <strong>and</strong> extraction.
After collection, queens were partially
narcotized to reduce body movements <strong>and</strong> the
possibility <strong>of</strong> releasing pheromone. Heads <strong>of</strong>
queens were separated using fine scissors, then
a small opening was made on the cuticle <strong>of</strong>
each side in the area between the eye <strong>and</strong> the
articulation <strong>of</strong> the m<strong>and</strong>ible with a fine blade
(No. 11) fixed on a dissecting knife h<strong>and</strong>le
(No.4). The gl<strong>and</strong> was grasped by its duct with
fine-tipped forceps <strong>and</strong> removed intact. Gl<strong>and</strong>s
were immediately extracted in vials containing
500 III <strong>of</strong> methylene chloride <strong>and</strong> stored at -
20°C for behavioral bioassays <strong>and</strong> chemical
analysis.
To examine the possibility <strong>of</strong> differences
between madibular extract <strong>of</strong> NM <strong>and</strong> II
queens, a retinue bioassay was conducted
following the procedure <strong>of</strong> Kaminski et al.
(1990). Bioassay arenas were prepared using 9-
cm diameter plastic Petri dishes. Young
workers aged 1-5 days were individually
selected from the brood frames <strong>of</strong> honey bee
colonies headed by A.m. queens. Upon
collection, workers were temporarily frozen
<strong>and</strong> 10 were placed in each bioassay arena.
Pseudoqueens were prepared from glass rods
(7-cm length X 0.6-cm diameter) with one end
heated for smooth surface to receive the
pheromone sample. The optimal amount <strong>of</strong>
extract for bioassay was determined by
preliminary tests <strong>of</strong> retinue behavior elicited by
0.004, 0.003, <strong>and</strong> 0.002 queen equivalents <strong>of</strong> QM
extracts (unpublished data). The lowest
concentration that could elicit a clear response
was found to be 0.002 Qeq (3 Ill). The 3 III was
applied on the smoothed tip <strong>of</strong> the glass rod
using a microsyringe <strong>and</strong> the solvent was
allowed to evaporate before introducing the
pseudoqueen to the queenless workers.
A total <strong>of</strong> 28-replicates (dishes) for each
treatment were tested. The response <strong>of</strong> workers
to the introduced pseudoqueens was
videotaped under red light in a temperaturecontrolled
room (25°C) for 15 minutes to allow

4COOOOO!
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!
Fig 1. Mean numbers <strong>of</strong> workers within a length <strong>of</strong> one
worker around m<strong>and</strong>ibular extracts <strong>of</strong> NM <strong>and</strong> II queens
collected after I <strong>and</strong> 2 weeks <strong>of</strong> oviposition during Spring
season <strong>of</strong> 2000. Bars marked by the same letter are not
significantly different.
time for differences in worker's response to NM
<strong>and</strong> II extracts to be observed. Response <strong>of</strong>
workers to solvent controls was also recorded.
Control data were not included in the statistical
tests due to small number <strong>of</strong> replicates.
Workers were used only once for each bioassay
<strong>and</strong> then killed by freezing. On playback, the
videotape was paused every 30 seconds <strong>and</strong> the
number <strong>of</strong> workers that were within one
worker length <strong>of</strong> the rod tip was recorded.
Total number <strong>of</strong> workers were divided by 30 to
obtain the mean number <strong>of</strong> workers found
around the rod for every 30 second.
Pentadecanoic acid (10 Ilg in 5 IIIsolvent)
was added as an internal st<strong>and</strong>ard to each <strong>of</strong>
the 500 III QM extracts, which were then
derivatized with diazomethane to convert fatty
acids to their methyl esters. Two-microliter
aliquots <strong>of</strong> each derivitized extract was injected
with a one III solvent plug in splitless mode
onto a 30-m x 0.25 mm HP-5MS capillary
column (Agilent Technologies) on Agilent
Technologies 6890 gas chromatograph
interfaced to 5973 N mass selective detector
(GC/MS). Analysis conditions were: injector
230 oC, oven 50-240 oC @ 15 OCI min. with a
flow <strong>of</strong> 1.2 ml/min helium <strong>and</strong> the flame
ionization detector (FlD) was set at 250 oc.
Fig 2. Representative Fill chromatogram for m<strong>and</strong>ibular
gl<strong>and</strong> extracts <strong>of</strong> NM <strong>and</strong> II queens collected after
I <strong>and</strong> 2 weeks <strong>of</strong> oviposition in Spring 2000.
*Arrows indicate peaks <strong>of</strong> significant differences: 9.90, 9-
keto-(E)-2-decenoic acid (9-0DA. high levels in NM
queens); 13.60, (Z)-9-octadecenoic acid (OLA, high levels
in II queens). QMP components, methyl p-hydroxybenzoate
(HOB) <strong>and</strong> (9-hydroxy-(E)-2-decenoic acid (9-HDA) are
shown at 8.02 <strong>and</strong> 9.93 m in respectively.
Chemical ionization FID chromatograms were
obtained. Electron ionization (EI) spectra were
also obtained for selected samples from all
treatments. The amount <strong>of</strong> each peak
(compound) was calculated based on the
internal st<strong>and</strong>ard in each sample. To identify
significant peaks <strong>and</strong> the QMP components in
the extracts, relative retention time <strong>and</strong> EI mass
spectra were compared to those <strong>of</strong> pure
compounds accessed via the EI library <strong>and</strong> to
authentic QMP samples obtained from
Pherotech® (Canada).
Minitab statistical s<strong>of</strong>tware (Version 13.1,
State College, Pennsylvania) was used to
compare responses to treatments. one-,two-.,
<strong>and</strong> three-way analysis <strong>of</strong> variance (ANoV A)
tests were used for the statistical analyses <strong>of</strong>
both behavioral bioassays <strong>and</strong> chemical
analysis.
Behavioral Bioassays:
M<strong>and</strong>ibular extracts for NM <strong>and</strong> II queens
1 <strong>and</strong> 2-weeks after oviposition were bioassyed.
Two-way ANoV A revealed that NM extracts
evoked higher responses than II extracts (df-87,

Table 1. Proportions <strong>of</strong> m<strong>and</strong>ibular gl<strong>and</strong> pheromone components in NM <strong>and</strong> II queens collected after I <strong>and</strong> 2 weeks <strong>of</strong> oviposition
during Spring <strong>of</strong> 2000.
Treatment 9-0DA 9-HAD HOB
NMWI 0.75 ±0.01" 0.75 ± 0.01" 0.05 ±0.02
NMW2 0.65 ± 0.03 0.31 ± 0.03 0.04 ± 0.02
lIWl 0.62 ±0.02 0.33 ± 0.03 0.06 ±0.01
IIW2 0.67 ± 0.03 0.28 ±0.04 0.05 ±0.02
F=4.75, P=0.032) with no effect for week (df=87,
F=0.55, P=0.460) or interaction between week
<strong>and</strong> mating type (df=87, F=1.54, P=0.219) (Fig.
1).
Chemical analysis <strong>of</strong> m<strong>and</strong>ibular
extracts:
M<strong>and</strong>ibular gl<strong>and</strong>s <strong>of</strong> NM <strong>and</strong> II queens
collected in Spring <strong>of</strong> 2000 after 1 <strong>and</strong> 2-weeks
<strong>of</strong> oviposition were analyzed (Fig. 2). All QMP
components were present except HVA, which
was below detectable limits. Two-way ANOV A
showed significantly higher amount <strong>of</strong> the
QMP major component 9-keto-(E)-2-decenoic
acid (9-0DA) in NM queens than in II queens
(df=27, F=4.8, P,=0.038) with no effect for week
(df=27, F=O.OO, P=0.964) <strong>and</strong> significant
interaction between week <strong>and</strong> mating type
(df=27, F=5.22, P=0.032). NM m<strong>and</strong>ibular
gl<strong>and</strong>s contained 79 ± 6.1 Ilg <strong>of</strong> 9-0DA whereas
II gl<strong>and</strong>s contained 62 ± 5.5 Ilg. The other QMP
components identified are HOB at retention
time 8.02 minutes <strong>and</strong> ± 9-HDA at 9.93 min. II
queens exceeded NM queens in one compound
that was non-QMP component identified as (Z)-
9-octadecenoic acid (oleic acid, OLA). II
contained 12.4 ±1.3 Ilg, NM contained 8.6 ± 1.2
Ilg (df=27, F=4.44, P=0.046). Levels <strong>of</strong> QMP
components were not affected by egg-laying
period.
Proportions <strong>of</strong> QMP:
Proportions <strong>of</strong> 9-0DA, ±9-HDA, <strong>and</strong> HOB
to the total amount <strong>of</strong> the QMP were compared.
Significant differences were found in
proportions <strong>of</strong> 9-0DA <strong>and</strong> ± 9-HDA <strong>of</strong> NM
queens after one week <strong>of</strong> oviposition (df=27,
F=4.76, 3.89, P=0.017, 0.011 respectively) (Table
1). Proportions <strong>of</strong> 9-0DA <strong>and</strong> ± 9-HDA
expressed age-dependent changes at each
period <strong>of</strong> ovipositiion.
Discussion
Chemical identification <strong>of</strong> gl<strong>and</strong>ular
extracts should be paired with quantitative
bioassays to evaluate the significance <strong>of</strong>
differences in their chemistry (Kaminski et al.,
1990). It has been well established that the
m<strong>and</strong>ibular gl<strong>and</strong> <strong>of</strong> honeybee queen secrets a
pheromone <strong>of</strong> the components that is-among
other behavioral <strong>and</strong> physiological effectscapable
<strong>of</strong> attracting worker bees to form a
retinue around the queen that includes
antennnating <strong>and</strong> licking the queen (Gary, 1961;
Pain, 1973; Winston, 1987; Slessor et al., 1988).
Prior to the chemical analysis <strong>of</strong> NM <strong>and</strong> II
queen m<strong>and</strong>ibular extracts, the retinue
response bioassay was conducted using glass
pseudo-queens treated with the pheromone.
This bioassay was designed to quantify shortrange
communication between worker bees <strong>and</strong>
glass pseudo-queens treated with pheromone
in laboratory conditions (Kaminski et al., 1990).
Our results showed the NM queen extracts
evoked stronger response in workers that II
extracts. This greater response to NM extracts
was mostly due to the high levels <strong>of</strong> QMP
major component, 9-0DA, they contained. II
queen extracts had higher levels <strong>of</strong> oleic acid
(Z)-9-octadecenoic acid (OLA). Oleic acid is
rarely reported among queen m<strong>and</strong>ibular gl<strong>and</strong>
secretions specially laying mated queens. The

presence <strong>of</strong> oleic acid in our NM <strong>and</strong> 11 samples
is in accordance with Engels et al., (1997) where
it was found in levels up to 25 Ilg in virgin
queen m<strong>and</strong>ibular gl<strong>and</strong>s <strong>and</strong> decreased only
after egg-laying initiation. Our 11 queens
contained 12.4 ± 1.3 Ilg whereas NM queens
contained 8.6 ± 1.2 Ilg <strong>of</strong> oleic acid. It was
possibly present in the m<strong>and</strong>ibular gl<strong>and</strong> as a
byproduct in the biosynthetic pathway for fatty
acids (9-0DA <strong>and</strong> ± 9-HDA) production. The
starting point for this biosynthetic pathway was
found to be octadecanoic acid (Plettner et al.,
1996). High levels <strong>of</strong> OLA accompanied by low
levels <strong>of</strong> 9-0DA in our 11 queen m<strong>and</strong>ibular
gl<strong>and</strong>s (vise versa in NM queens) strongly
indicate slower rate <strong>of</strong> fatty acids production in
11 queens m<strong>and</strong>ibular gl<strong>and</strong>s compared to NM
queens. This possible slower rate <strong>of</strong> fatty acids
biosynthesis in 11 queens might be correlated to
the lower survival rates in 11 queens reported
by several authors (Robert, 1946; Herbo <strong>and</strong>
Szabo, 1984; Konopacka, 1987; Wilde <strong>and</strong> Loc,
1997). Other studies reported oleic acid as the
major compound in the tergal gl<strong>and</strong> secreation
<strong>of</strong> honeybee queens (Wossler <strong>and</strong> Crewe,
1999a). Tergal secretions are believed to
maintain <strong>and</strong> stabilize the retinue behavior
around the queen through contact
chemoreception after its formation by
m<strong>and</strong>ibular secretions that attract workers
across a distance (Velthuis, 1972, 1985; Vierling
<strong>and</strong> Renner, 1977). The common presence <strong>of</strong>
OLA in m<strong>and</strong>ibular <strong>and</strong> tergal gl<strong>and</strong> secretions
indicates the necessity <strong>of</strong> behavioral <strong>and</strong>
chemical studies to examine possible roles for
this compound in the honeybee communication.
Effects <strong>of</strong> egg-laying period on levels <strong>of</strong>
m<strong>and</strong>ibular secretions did not show significant
changes in QMP levels at 1 <strong>and</strong> 2 weeks <strong>of</strong>
oviposition. Lack <strong>of</strong> correlation between QMP
composition <strong>and</strong> ovarian development was
previously reported by Plettner et al., (1993).
Proportions <strong>of</strong> QMP components identified
were compared <strong>and</strong> revealed significant
changes in proportions <strong>of</strong> NM queens only. NM
queens <strong>of</strong> one week oviposition period had
significantly higher levels <strong>of</strong> 9-0DA <strong>and</strong> lower
levels <strong>of</strong> ± 9-HDA. Queens mate naturally with
several drones during mating flight (Winston,
1987) which might causes variability in the
amount <strong>of</strong> semen stored in the spermatheca
(Woyke, 1966), whereas a uniform doses <strong>of</strong>
semen were given to our 11 queens. Cobey
(1999) found NM queens to have high
variability in brood production compared to 11
queens inseminated with 8 ~ll<strong>of</strong> semen. Filling
<strong>of</strong> the spermatheca affects capability <strong>of</strong> egg
laying in queens (Harbo, 1971). Workers can
detect small changes in queen signal resulted
from egg laying activity <strong>and</strong> sperm conditions
in the spermatheca (Butler, 1957; Winston,
1987).
In each group <strong>of</strong> queens, a pattern <strong>of</strong>
ontogenetic changes in the QMP proportions <strong>of</strong>
NM <strong>and</strong> 11 queen m<strong>and</strong>ibular gl<strong>and</strong>s at each
oviposition period was found. This indicates a
biosynthetic change in the m<strong>and</strong>ibular gl<strong>and</strong>s
associated with age progression <strong>and</strong> egg laying
activity. Results presented that 9-0DA
proportions <strong>of</strong> NM queens at week one
oviposition period were significantly higher
than other 9-0DA proportiions <strong>of</strong> NM <strong>and</strong> 11
queens. This was accompanied by the
significantly lowest proportion <strong>of</strong> 9-HDA in
the same group <strong>of</strong> queens. Crewe (1982) <strong>and</strong>
Slessor et al., (1990) reported ontogenetic
patterns <strong>of</strong> QMP components. Biosynthetic
change in the m<strong>and</strong>ibular gl<strong>and</strong> after queen
mating that resulted in higher proportions <strong>of</strong> 9-
HDA, HOB, <strong>and</strong> HVA accompanied by
decreased proportions <strong>of</strong> 9-0DA was reported
by Pankiw et al., (1996). Engels et al., (1997)
suggested three ontogenetic patterns for queen
m<strong>and</strong>ibular secretions: premating pattern <strong>of</strong>
newly emerged virgins with dominant OLA
(oleic acid), mating pattern <strong>of</strong> receptive virgin
consisting <strong>of</strong> 9-0DA, OLA <strong>and</strong> small amounts
<strong>of</strong> 9-HDA, <strong>and</strong> postmating pattern <strong>of</strong> dominant
queens with high amounts <strong>of</strong> 9-0DA, medium

proportions <strong>of</strong> (9-HDA, less aLA, <strong>and</strong> small
amounts HOB <strong>and</strong> HVA. Most <strong>of</strong> our NM <strong>and</strong>
II queens approximately fit in the postmating
pattern.
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