Studies were conducted on the physiological mode of action and mode of perception of a queen pheromone that inhibits dealation (wing-shedding) and ovary development in virgin queens of the fire ant Solenopsis inuicta. Winged virgin queens were removed from the pheromonal signal (queen) to compare the response time for dealation, a behavioral response, and ovary development, a physiologi- cal response. Dealation was always accompanied by some degree of ovary development, whereas some individuals exhibited slightly developed ovaries before dealation occurred, suggesting that ovary development precedes dealation by several hours to a day. The response time of virgin queens following extinction of the pheromonal signal was highly variable and was related to colony source and body weight. Individuals from monogyne (single functional queen) colonies were more responsive than those from polygyne (multiple functional queens) colonies, a result consistent with the high cumulative levels of queen pheromone in the latter colonies. Virgin queens treated topically with the juvenile hormone (JH) analogue, methoprene, dealated and developed their ovaries in the presence of the pheromone (queen), suggesting that the pheromonal mode of action involves the suppression of JH titers. To obtain more precise information on the physiological processes underlying the inhibition of ovary develop- ment, vitellogenin titers were determined for virgin queens and functional (egg-laying) queens. Despite having undeveloped ovaries, virgin queens had vitellogenin titers that were as elevated as those of functional queens. This suggests that the effect of the low JH titers resulting from the primer pheromone is on the uptake of vitellogenin by the oocytes rather than on vitellogenin synthesis. The possible mode of perception of the inhibitory pheromone was also investigated. Virgin queens whose antennae had been removed dealated in the presence of the queen, suggesting that the pheromone acts by stimulating sensory cells in the antennae. The results are incorporated into a general model for the mode of action of the queen primer pheromone.

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Pergamon 0022-1910(94)E0013-6

J. fns~ct Phwiol. Vol. 40, No. 7, pp. 601-610. 1994

Copyright :C 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved

0022-1910/94 $7.00 + 0.00

Studies on the Mode of Action of a Queen

Primer Pheromone of the Fire Ant

Solenopsis invicta

EDWARD L. VARGO,* MICHELE LAUREL*

Received 5 October 1993: reaised 16 December 1993

Studies were conducted on the physiological mode of action and mode of perception of a queen

pheromone that inhibits dealation (wing-shedding) and ovary development in virgin queens of the fire

ant Solenopsis inuicta. Winged virgin queens were removed from the pheromonal signal (queen) to

compare the response time for dealation, a behavioral response, and ovary development, a physiologi-

cal response. Dealation was always accompanied by some degree of ovary development, whereas some

individuals exhibited slightly developed ovaries before dealation occurred, suggesting that ovary

development precedes dealation by several hours to a day. The response time of virgin queens following

extinction of the pheromonal signal was highly variable and was related to colony source and body

weight. Individuals from monogyne (single functional queen) colonies were more responsive than those

from polygyne (multiple functional queens) colonies, a result consistent with the high cumulative levels

of queen pheromone in the latter colonies. Virgin queens treated topically with the juvenile hormone

(JH) analogue, methoprene, dealated and developed their ovaries in the presence of the pheromone

(queen), suggesting that the pheromonal mode of action involves the suppression of JH titers. To obtain

more precise information on the physiological processes underlying the inhibition of ovary develop-

ment, vitellogenin titers were determined for virgin queens and functional (egg-laying) queens. Despite

having undeveloped ovaries, virgin queens had vitellogenin titers that were as elevated as those of

functional queens. This suggests that the effect of the low JH titers resulting from the primer

pheromone is on the uptake of vitellogenin by the oocytes rather than on vitellogenin synthesis. The

possible mode of perception of the inhibitory pheromone was also investigated. Virgin queens whose

antennae had been removed dealated in the presence of the queen, suggesting that the pheromone acts

by stimulating sensory cells in the antennae. The results are incorporated into a general model for

the mode of action of the queen primer pheromone.

Formicidae Reproduction Primer pheromone Queen Juvenile hormone

INTRODUCTION

The study of insect chemical communication is a

rapidly expanding field. The last 30 years have seen

tremendous progress in our understanding of the chem-

istry of insect pheromones, their mode of perception,

and the regulation of their biosynthesis and release

(Mayer and Mankin, 1985; Tamaki, 1985; Kaissling,

1986; Prestwich and Blomquist, 1987; Mayer and

McLaughlin, 199 1; Schneider, 1992; Raina and

Menn, 1993). However, nearly all of this progress has

concerned releaser pheromones, i.e. intraspecific chemi-

cal signals that elicit behavioral responses, such as

sex attractants. In contrast, progress has been much

slower in our understanding of primer pheromones,

*Department of Zoology, University

U.S.A. of Texas, Austin, TX 78712,

which have less rapid but more profound effects on the

physiology of the target individuals (Wilson and Bossert,

1963).

Primer pheromones are thought to be widespread

among the social insects. They are produced by the

primary reproductives (queens in the social Hymen-

optera) and serve to regulate ovary development in

adult female members of the colony and influence the

developmental fate of larvae (Wilson, 1971). We cur-

rently have little information on the chemical nature of

social insect queen primer pheromones, their mode of

perception, or their physiological mode of action. The

only social insect primer pheromone that has been

identified is that of the queen honey bee, which orig-

inates primarily in the mandibular gland [reviewed in

Winston and Slessor (1992)]. Originally, this queen

mandibular pheromone, especially its major constituent,

(E)-9-keto-2-decenoic acid, was reported to inhibit both

601

602 EDWARD L. VARGO and MICHELE LAUREL

the rearing of new queens (Butler, 1959a, 1960, 1961;

Butler and Paton, 1962; Butler and Callow, 1968) and

ovary development in workers (de Groot and Voogd,

1954; Butler 1959b; Butler et al., 1962; Butler and Fairey,

1963). However, recent studies using synthetic com-

ponents in natural proportions suggest that, although

this pheromone complex inhibits the rearing of new

queens (Winston et al., 1989, 1990), it does not inhibit

worker ovary development (Willis et al., 1990).

There are few data concerning the mode of perception

of the honey bee queen pheromone. Kaissling and

Renner (1968) found that (E)-9-keto-2-decenoic acid

elicits firing by specialized sensory cells in the antennae

of worker honey bees. However, the response of worker

antennae to the other components of the queen

mandibular gland secretion has not been investigated.

Information is also lacking concerning the physiological

mode of action of the honey bee queen pheromones.

Kaatz et al. (1992) recently found that (E)-Pketo-2-

decenoic acid inhibits juvenile hormone (JH) secretion

by the corpora allata in young workers. The conse-

quences of lower JH synthesis in response to (E)-9-keto-

2-decenoic acid in young bees are unclear, however,

because this pheromone component may not inhibit

ovary development (Willis et al., 1990). Furthermore,

developed ovaries in queen and worker honey bees is not

related to elevated JH titers (Robinson et al., 1991,

1992), suggesting that in honey bees JH does not play

the role it typically assumes in regulating insect ovary

development (Koeppe et al., 1985).

In the bumble bee, Bombus terrestris, ovary develop-

ment appears to follow the more general insect scheme

involving regulation by JH (Riiseler, 1977; Riiseler and

Rdseler, 1978). In this species queens produce a phero-

mone of known composition in the mandibular glands

that inhibits ovary development in workers by suppress-

ing JH synthesis (Riiseler et al., 1981). No information

is avaiIable on the possible mode of transmission or

perception of this bumble bee queen pheromone.

Several effects of queen pheromones have been docu-

mented in the fire ant Solenopsis invicta. These include

the development of male and female sexuals (Vargo and

Fletcher, 1986), dealation (wing shedding) and ovary

development in winged virgin queens (Fletcher and

Blum, 1981a), and ovary development among cohabiting

egg-laying queens in polygyne (multiple-queen) colonies

(Vargo, 1992). Although there is little specific infor-

mation available on the mode of action of these phero-

mones, evidence to date suggests that pheromonal

regulation of ovary development involves JH.

Results of studies in which virgin queens in isolation

were treated topically with synthetic JH, either by itself

(Kearney et al., 1977) or in combination with allatec-

tomy (Barker, 1978, 1979), indicate that both dealation

and ovary development are under the control of JH.

Using topical application of the JH analogue, metho-

prene, Vargo (1992) obtained evidence that the rate of

oviposition among queens in polygyne colonies is regu-

lated by JH. Fletcher and Blum (1983) and Vargo (1992)

hypothesized that fire ant queen primer pheromones

prevent ovary development in winged queens and repro-

ductively active queens, respectively, by inhibiting JH

synthesis. However, the possible JH-suppressing effect of

the pheromone has not been tested.

In this paper, we investigate several aspects of the

mode of action and mode of perception of the phero-

mone inhibiting dealation and ovary development in

S. invicta virgin queens. Specifically, we address four

questions. First, how closely linked are the responses of

ovary development and dealation? These two responses

are very different; dealation is a behavioral response in

which an individual breaks off her wings with the hind

legs, whereas ovary development is a physiological re-

sponse. Although these two responses occur in individ-

uals removed from the influence of the queen, it is not

known whether they occur simultaneously or whether

there is a delay separating them. Second, can the inhibi-

tory effects of the pheromone be overcome with topical

application of a JH analogue? Third, is ovary develop-

ment in virgin queens blocked at the vitellogenin-

synthesis or vitellogenin-uptake stage? Finally, are

antenna1 receptors in the virgin queens involved in the

perception of the pheromone? Answers to these ques-

tions give a more complete picture of the physiological

mode of action of this queen primer pheromone.

MATERIALS AND METHODS

Source and maintenance of ants

All ants used in these experiments originated from

Travis and Hays Counties, TX. The colonies were

collected by excavation of the mound, and the ants were

removed from the soil by flooding (Jouvenaz et al.,

1977). Dates of collection are given below. Colonies were

housed in plastic trays (40 x 52 x 8 cm) equipped with

four nests (14 cm dia Petri dishes half-filled with damp

plaster) and maintained in the laboratory at 29 + 2°C

and natural photoperiod. The ants were fed crickets

(Acheta domesticus) daily and given sugar water and tap

water ad libitum.

Timing of dealatdon and ovary development

Monogyne colonies (n = 9) used in this experiment

were collected on 6 May 1991 and polygyne colonies

(n = 9) were collected on 13 May 1991. All colonies had

numerous winged virgin queens present at the time of

collection.

Dealation and ovary development were studied using

two winged virgin queens placed in small colony frag-

ments (2.5 ml workers and brood) as described by

Fletcher and Blum (1981 b). Virgin queens were moni-

tored every 12 h for dealation, defined as having lost at

least three of the four wings. The time elapsed for

dealation to occur in at least one of the two virgin queens

present in each fragment was recorded. Ovary develop-

ment was assessed by dissecting virgin queens in 70%

ethanol under a dissecting microscope and counting the

FIRE ANT PRIMER PHEROMONE 603

total number of chorionated eggs (fully-formed eggs, i.e.

those 20.4 mm wide x 0.28 mm long) present in the

ovarioles and common oviduct. At the beginning of each

experiment, a sample of virgin queens from each colony

was weighed and dissected to assess ovary development.

In this experiment, the timing of dealation and ovary

development was investigated in virgin queens originat-

ing from monogyne and polygyne colonies as well as the

effect of the weights of individual queens on response

time. Ten virgin queens from each colony were weighed

and then dissected to assess ovary development. Ten

others were weighed and placed in pairs into small

colony fragments (n = 5 fragments per colony); each

individual was marked with one of two colors of paint

(Tex Pen"', Mark-Tex Corp., Engelwood, NJ) for later

identification. The fragments were examined every 12 h

for the presence of dealates. As soon as at least one

dealate was observed in a fragment, both virgin queens

present were removed, dissected and their ovary develop-

ment assessed. The experiment ran for three weeks, after

which time all remaining virgin queens were dissected to

determine the degree of ovary development.

Methoprene treatment

The ants used for this experiment were of the mono-

gyne form and were collected 26 and 30 May 1992 from

Hays and Travis Counties, TX. All colonies contained

numerous alate queens. Approximately one month after

collection, the colonies were sieved to remove all sexual

forms [adults, pupae and large fourth instar larvae;

Vargo (1988)] and reduced to a size of 10 g adult workers

and brood (approx. 5500 adult workers, 4000 worker

pupae and 8500 larvae of all stages). The colonies were

housed in rearing trays (40 x 52 x 8 cm), each contain-

ing a single nest as described above. The mother queen

of the colony was placed in the fragment. Twenty virgin

queens, all field-reared, were returned to each colony.

Half of these were topically treated with 0.5 pg metho-

prene in 0.5 ~1 acetone by placing the solution on the

abdominal dorsum. The other half of the virgin queens

was treated with only the acetone carrier. Each of the

queens was marked with a spot of Texpen ink identifying

it to group. The colony fragments were monitored daily

for three days for the presence of any dealated virgin

queens. All dealates present on a given day were re-

moved and dissected to assess ovary development. At the

end of three days, all remaining winged virgin queens

were removed and their ovaries examined.

Vitellogenin titers

Vitellogenin titers were determined by SDS-PAGE

based on the methods of Martinez and Wheeler

(1991a, b). Hemolymph from individual queens was

collected in 0.5 ~1 capillary tubes through a small in-

cision on the thoracic dorsum and immediately trans-

ferred into a tube containing 10 ~1 SDS sample buffer

(62 mM Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mer-

captoethanol, 2% SDS, 0.005% Bromophenol Blue).

Freshly-laid eggs were homogenized directly into 10 ~1

SDS sample buffer. Samples were centrifuged for 3 s at

8000g and stored at -20°C. Just prior to loading, the

samples were thawed, boiled for 5 min, centrifuged for

3 s at SOOOg, and transferred to ice. Aliquots from each

sample were used for SDS-PAGE analyses.

Samples were subjected to SDS-PAGE using a 7%

polyacrylamide gel with a 4% stacking gel. Electrophor-

esis was performed at constant voltage with a starting

current of 20 mA. Gels were stained with 0.24%

Coomassie Brilliant Blue R 250 in 45.6% methanol,

9.2% acetic acid and destained in 7.5% acetic acid, 5%

methanol.

The density of the vitellogenin bands was quantified

for each sample using a video densitometer designed by

L. L. Poulsen (U.S. Patent No. 5194949). The band

corresponding to the most abundant protein in freshly-

laid eggs (approx. 180 kDa) was determined for each

sample. The quantity of vitellogenin present was deter-

mined by comparison with the density of a known

quantity of myosin standard (200 kDa).

All queens sampled were dissected to assess ovary

development. The ovaries were removed and the maxi-

mum number of opaque vitellogenic oocytes per ovariole

was determined (Tschinkel, 1988) by examining several

ovarioles.

Antennectomy

Five monogyne colonies, containing numerous winged

queens, were collected 26 and 30 May 1992. Approxi-

mately four weeks after collection, the colonies were

sieved to remove sexuals, and the workers and brood

were divided into two equal halves based on weight

(mean + SD weight of colony halves = 11.5 + 2.8 g). The

mother queen was returned to one of the halves (queen-

right halves). Each colony half was given a total of 20

nestmate winged virgin queens, all of which were field

reared and at least one month post emergence age. The

antennae were removed from 10 of the virgin queens in

each colony half by cutting the scape near the insertion

point with fine dissecting scissors. The 10 control virgin

queens in each colony half were amputated at a middle

leg by cutting the proximal region of the femur. The

colony halves were monitored daily for the presence of

dealates. Any dealates observed were removed and dis-

sected to assess ovary development as above. Ovary

development was also assessed for 10 winged virgin

queens from each colony at the time of colony division.

The experiment was terminated after 15 days.

RESULTS

Timing of dealation and ovary development

There was a large difference between monogyne and

polygyne colonies in the frequency of virgin queen

dealation; by 72 h dealation had occurred in all

25 fragments containing virgin queens from mono-

gyne colonies, whereas dealation had occurred in only

three (12%) of the 25 fragments from polygyne colonies

(X: = 25.1, P < 0.0001). The number of polygyne

604 EDWARD L. VARGO and MICHELE LAUREL

fragments containing dealates had risen to only 7 (28%)

by the end of the experiment. Virgin queens from

polygyne colonies that dealated took nearly four times

longer to do so than did those from monogyne colonies

(mean = 120.0 + 156.0 and 31.7 + 15.8 h for polygyne

and monogyne, respectively; t,, = 2.90, P < 0.01, two-

tailed test). Among the monogyne colonies, there was a

significant effect of colony on the time to dealation

(F4.20 = 4.49, P < 0.01).

Dealation was related to both higher weights and

increased ovary development. For the colony fragments

in which only one of the two virgin queens dealated, the

dealated individual had an initial weight that was on

average 4% more than the alate (mean differ-

ence = 0.58 4 0.85 mg; t,, = 3.22, P < 0.005, two-tailed

paired test). When the virgin queens from monogyne and

polygyne colonies were analyzed separately, the dealates

in the polygyne form had an initial weight that was 8.6%

greater than alates (mean difference = 1 .l + 0.9 mg,

t5 = 3.0, P < O.OS), whereas in the monogyne form

dealates weighed only 2.8% more (mean differ-

ence = 0.4 + 0.8 mg, t,, = 2.0, P > 0.05). The difference

in ovary development between the dealate and alate in

the same fragment was even more pronounced. When

virgin queens from monogyne and polygyne colonies

were taken together, the dealated females had an average

of 7.6 f 2.0 chorionated oocytes compared to 3.2 + 2.8

for the alates (tz, = 6.85, P < 0.0001, two-tailed paired

test). Again, when analyzed separately, this difference

was greater among females of the polygyne form, where

dealates had 6.2 f 2.0 more chorionated oocytes than

did alates (ts = 7.4, P < 0.001); in fragments from

monogyne colonies dealates had 3.8 f 3.2 more

(tls = 4.8, P < 0.0002).

A comparison of the degree of ovary development in

relation to dealation status was performed by means of

a two-way ANOVA, with social form and dealation

status as the independent variables. In order to detect

changes in ovary development under queenless con-

ditions, winged virgin queens from the queenright

colonies at the start of the experiment were also included

in this analysis. Both social form (F,,,9, = 75.4,

P < 0.0001) and dealation status (F2.,9, = 105.1,

P < 0.0001) had highly significant effects, and the inter-

action term was also significant (F2,19, = 3.1, P < 0.05).

Therefore, the effects of dealation status were analyzed

separately for each form. In both social forms, there was

a highly significant difference in the degree of ovary

development among all three groups, i.e. dealates and

alates in the queenless colony fragments and alate virgin

queens in queenright colonies at set-up (Table 1;

F2;.97 = 80.3, P < 0.0001 and F2,94 = 72.8, P < 0.0001, for

the monogyne and polygyne forms, respectively). In

both forms, dealates showed the most ovary develop-

ment, whereas alate virgin queens in the queenless

colony fragments had significantly more developed

ovaries than did those in the presence of the queen at the

start of the experiment (P < 0.05, Tukey test). These

results suggest that the responses of dealation and ovary

TABLE 1. Ovary development (mean k SD) of virgin

queens in response to queenlessness and dealation

Form/condition n

No.

chorionated

oocytes

Results of the

Tukey test

Monogyne

Q+ 50 2.1 + 2.1 A

Alate 16 4.3 +_ 2.4 B

Dealate 34 8.7 f 2.7 C

Pol.vgyne

Q+ 50 0.2 f 0.5 A

Alate 39 1.0* 1.2 B

Dealate 8 5.4 _+ 2.8 C

Queenright condition (Q +) refers to condition of the virgin

queens in their natal colonies at the time the experiment

was started. Virgin queens were set up in pairs in small

queenless colony fragments. As soon as at least one

individual in each fragment dealated, both virgin queens

present were dissected to assess ovary development.

Included in the polygyne form are those fragments in

which no dealation occurred during the 3-week exper-

iment. Treatments with different letters within each form

differed significantly (P < 0.05, Tukey test).

development are tightly linked, but that ovary develop-

ment begins slightly before dealation.

Methoprene treatment

Of the 50 methoprene treated virgin queens, 44 (88%)

individuals dealated by the end of the 3-day period

(Fig. 1), with a mean of 8.8 + 1.3 individuals per colony

100 y

0

Methoprene Control

FIGURE 1. Effect of topical treatment of methoprene (0.5 ng in 0.5 ~1

acetone; n = 50) on dealation and ovary development (mean & SE) in

the presence of queen inhibitory pheromone. Controls (n = 45) were

treated with 0.5 ~1 of the acetone carrier.

FIRE ANT PRIMER PHEROMONE 605

(range = 7-10). In contrast, none of the control virgin

queens shed their wings during this time. The difference

between the two groups was highly significant when

analyzed either by pooling individuals from all colonies

(P < 0.0001, Fisher exact test) or by examining each

colony separately (all P < 0.0025, Fisher exact test). A

total of five control individuals from three different

colonies were found dead of unknown causes.

Individuals in the methoprene treatment had far more

developed ovaries (Fig. 1). A two-way ANOVA showed

a significant effect of both treatment (F,,, = 74.6,

P < 0.0001) and colony (F4.85 = 3.2, P < 0.02) but not of

the interaction term (F4.85 = 1.3, P > 0.25).

Vitellogenin titer determinations

As shown in Fig. 2, there was no significant difference

among the three categories of queens in their titers of

vitellogenin (F2,2, = 0.6, P > 0.5). This is in sharp con-

trast to the large differences in ovary development

(Fig. 2; F2.2, = 221.5, P < O.OOOl), in which all categories

differed significantly from the others (P < 0.05, Tukey

test). Thus, inhibited virgin queens with undeveloped

ovaries had vitellogenin titers comparable to those of

40

s 30

a

s

.; 20

B

B

5 10

0

n .5! 20

b

P 15

s

ii

.g 10

g

al

.? 5

$

>i

r la

r" 0 WQ PQ MQ

FIGURE 2. Relationship between vitellogenin titers (pg/pl hemo-

lymph, expressed as myosin equivalents) and ovary development in S.

inuicta queens. Shown are means f SE. WQ, winged virgin queens in

the presence of a pheromone producing queen (n = IO); PQ, egg-laying

dealated queens from polygyne colonies (n = 9); MQ, egg-laying

dealated queens from monogyne colonies (n = 5). Groups with differ-

ent lower case letters differed significantly (P < 0.05, Tukey test). absence (P = 0.15, Fisher exact test).

FIGURE 3. Effect of removing antennae on dealation in the presence

and absence of queen inhibitory pheromone. The antennectomized

(-antennae) group (n = 9 and 25 in the presence and absence of the

queen, respectively) had their antennae removed by cutting them off

at the proximal end of the scape. The control group (n = 41 and 49 in

the presence and absence of the queen, respectively) had one middle

leg removed by cutting at the proximal end of the femur. A signifi-

cantly higher frequency of antennectomized individuals dealated in the

presence of the queen (P <O.OOl. Fisher exact test) but not in her

actively laying queens with highly developed ovaries.

These results suggest that the effect of the inhibitory

pheromone is to block ovary development primarily

by suppressing the uptake of vitellogenin rather than

affecting its rate of synthesis.

Antennectomy

There was considerable mortality of the virgin queens

whose antennae had been removed. Of the 50 treated

individuals, 25 (50%) died in the queenless colony halves

during the experiment (range = 3-9 per colony half). In

contrast only a single control individual died in the

queenless halves. Mortality was higher in the queenright

'halves, in which only nine of the antennaeless individuals

survived (range = 4-o survivors per colony half), as

opposed to 38 survivors in the control group. In some

cases, workers were seen attacking individuals, all of

which were wingless. It is not known whether dealation

occurred before attack by workers or as a result of

attack. However, since no alates were seen to be at-

tacked, it is likely that worker attack followed dealation

rather than causing it.

Among the surviving virgin queens, there was a strong

difference in the frequency of dealation in the queenright

halves but not the queenless halves (Fig. 3). In the

queenright halves, all-nine of the surviving ir tdividuals

20

0 - Antennae Control

606 EDWARD L. VARGO and MICHELE LAUREL

whose antennae had been removed dealated, whereas

only one (2.6%) of the controls shed her wings

(P < 0.001, Fisher exact test). In contrast, the frequency

of dealation in the two groups was similar in the

queenless halves (P = 0.145, Fisher exact test).

In comparing ovary development, individuals were

grouped according to whether queen pheromone was

present or absent (queenright vs queenless colony

halves), and whether they had dealated in the course of

the experiment (alate vs dealate). Out of the eight

possible groups (2 pheromone conditions x 2 treat-

ments x 2 states for wing presence), three groups were

not included because one group (antennaeless alates in

queenright colony halves) had no members and two

groups (antennaeless alates in queenless colony halves

and control dealates in queenright colony halves) had

very few members (n = 2 and 1, respectively). Individ-

uals that were in the queenright colonies on the day the

experiment started also were included in the analysis. An

ANOVA showed a significant difference among the

groups (& = 1.3, P < 0.0001). As seen in Table 2, this

difference was due to the greater degree of ovary devel-

opment in the control dealates in the queenless colony

halves than in those of all the other groups. Thus,

although antennectomy caused virgin queens to dealate,

even when in the presence of a pheromone producing

queen, development of the ovaries that normally accom-

panies wing shedding did not occur.

DISCUSSION

The finding that topical application of a JH analogue

to virgin queen fire ants overcomes the inhibitory effects

of the queen pheromone strongly suggests that the

pheromone inhibiting dealation and ovary development

acts by suppressing JH titers. The precise mechanism of

JH titer suppression was not investigated, but a likely

candidate is inhibition of JH synthesis by the corpora

allata. There is growing evidence from social insects that

queen primer pheromones affect JH titers by suppressing

the activity of the corpora allata. In the bumble bee,

B. terrestris, Roseler et al. (1981) found that extracts of

queen mandibular glands prevented ovary development

in workers by inhibiting JH production by the corpora

allata (Riiseler, 1977; Rijseler and Roseler, 1978). Re-

cently, Kaatz et al. (1992) showed in the honey bee that

synthetic (E)-9-oxo-2-decenoic acid, the principal com-

ponent of the honey bee queen mandibular gland se-

cretion, inhibits JH synthesis by the corpora allata in

young (8 day-old) workers. However, the effects of lower

rates of JH synthesis in young bees is not clear, because

JH does not appear to be the gonadotropic hormone in

this species (Robinson et al., 1991, 1992). In the termite,

Zootermopsis angusticollis, Greenberg and Tobe (1985)

reported that larvae had lower rates of JH synthesis by

the corpora allata in the presence of the king and queen

than in their absence. It is likely that a pheromone

produced by the royal pair is responsible for this effect,

but the existence of an inhibitory pheromone in

Z. angusticollis has not been conclusively demonstrated

(Stuart, 1979).

Although it is well established that social insect queens

inhibit ovary development in nestmates, little is known

about the process of oogenesis and the physiological

mechanisms underlying its control. The present results

demonstrate that virgin queens of S. invicta, whose

ovary development is blocked by the queen inhibitory

pheromone, have vitellogenin titers comparable to those

of egg-laying queens. Thus in fire ants, the presence of

vitellogenin in the hemolymph is not sufficient to induce

yolk deposition, a phenomenon noted in other insects

(Bell and Barth, 1971), including the honey bee (Engels,

1974) and the ant Camponotus festinatus (Martinez and

Wheeler, 1991a). Evidently, in many insects a specific

trigger is necessary for vitellogenin uptake to occur, and

it is such a trigger that is blocked by the fire ant queen

inhibitory pheromone. As discussed below, the trigger in

S. invicta queens may be a relatively high titer of JH.

Although the main effect on ovary development appears

to be inhibition of oogenesis, it is nevertheless possible

that the queen pheromone reduces the rate of vitel-

logenin synthesis in inhibited virgin queens compared to

reproductively active queens. However, because vitel-

logenin is not incorporated in the former group, it

accumulates and eventually reaches titers as elevated as

those in egg laying queens, which may be synthesizing

TABLE 2. Effect of antennectomy and queen pheromone on ovary development in virgin

queens

No. chorionated Results of

oocytes Tukey test

Treatment Dealated n (mean * SD) (P < 0.05)

Queenright colony at start _ 50 1.5 f 2.4 B

Queenright colony half

Antennae removed + 9 1.2k2.9 B

Control _ 37 0.9 * 1.5 B

Queenless colony half

Antennae removed + 23 1.9k2.6 B

Control - 9 2.6 & 3.6 B

Control + 40 1.3 & 4.6 A

Treatments with different letters differed significantly (P < 0.05. Tukey test).

FIRE ANT PRIMER PHEROMONE 607

vitellogenin at higher rates. Studies of the rate of

vitellogenin synthesis and turnover in inhibited and

reproductively active queens are needed to determine

possible effects of the pheromone on vitellogenin

synthesis.

In this study, we found that virgin queens amputated

of their antennae dealated in the presence of a queen

producing the primer pheromone. This result is expected

if the primer pheromone is perceived through sensory

cells in the antennae, and the inhibitory signal is then

transmitted via the nervous system to the target tissues,

presumably the corpora allata. In many insects, the

activity of the corpora allata is regulated by neuro-

secretory cells in the brain [reviewed in Feyereisen

(1985)]. Results from extirpation experiments on

S. inuicta virgin queens (Barker, 1978) are consistent

with this general model and suggest that the median

neurosecretory cells regulate the activity of the corpora

allata. In line with the present results, Sorensen et al.

(1985) found that the pheromone components, which

appear to be relatively non volatile (Fletcher and Blum,

198la), could be quickly and efficiently transported

throughout fire ant colonies via surface contact.

A puzzling result of the antennectomy experiment was

that ovary development in amputated individuals did

not coincide with dealation. This is unexpected if inhi-

bition of both dealation and ovary development is

achieved simply by pheromonal stimulation of sensory

cells in the antennae, which in turn act on the corpora

allata to inhibit JH synthesis. The reasons for the

uncoupling of dealation and ovary development in this

case are not clear, but possible explanations include:

(1) the presence of other sensory cues perceived

by the antennae that are needed to stimulate

oogenesis; and

(2) antennectomy induces dealation by affecting the

nervous system via some alternate route not

involving JH.

We do not yet have sufficient data to exclude either of

these possibilities.

Based on the results of the present study, we propose

a general model, illustrated in Fig. 4, for the mode of

action of the fire ant queen pheromone that inhibits

dealation and ovary development. The pheromone is

perceived by sensory cells in the antennae. Stimulation

of these sensory cells inhibits the brain from releasing

chemical and/or neural signals that stimulate the cor-

pora allata to synthesize JH. Consequently, the JH titer

is maintained at relatively low levels in the presence of

the pheromone. While the JH titer is kept relatively low,

dealation and oogenesis by virgin queens are prevented.

Presumably, JH acts directly on the nervous system to

elicit dealation, whereas it acts indirectly on ovary

development by stimulating the oocytes to absorb pre-

viously synthesized vitellogenin. The uncoupling of

dealation and ovary development following antennec-

tomy, as discussed above, suggests the physiological

control of these processes is more complicated than

indicated here, but the precise nature of these compli-

cations has yet to be determined.

We also hypothesize that JH controls vitellogenin

synthesis by the fat body, as occurs in many insects

(Koeppe et al., 1985), but that vitellogenin synthesis has

a lower JH threshold than does vitellogenin uptake by

the ovaries. Thus the pheromone does not completely

suppress JH synthesis, but reduces it to a low level,

keeping the JH titer below the vitellogenin absorption

threshold but above the vitellogenin synthesis threshold.

Further studies are needed to test and refine this model.

Of critical importance in this regard will be the identifi-

cation of the active pheromone components and JH titer

determinations.

Once removed from the queen primer pheromone,

ovary development in virgin queens appears to begin

slightly ahead of dealation. Although several studies

have shown that dealation by virgin queens of S. invicta

is associated with eventual oogenesis and oviposition

(Kearney et al., 1977; Barker, 1978; Tschinkel and

Howard, 1978; Fletcher and Blum, 198la. 1983; Fletcher

et al., 1983) the precise timing of these events was

previously unknown. Fletcher and Blum (1981a) dis-

sected dealate virgin queens one week after queenlessness

and found some with developed ovaries. Fletcher et al.

(1983) reported that some dealate virgin queens exam-

ined 72 h after queenlessness exhibited ovary develop-

ment. The present study, in which virgin queens were

dissected within 12 h post-dealation, offers a more de-

tailed view of the timing. All of the dealates showed

some signs of ovary development, whereas only some of

the alates, which had been free from the queen phero-

mone for the same period of time as the dealates,

exhibited signs of ovary development. The finding that

ovary development appears to precede dealation

suggests that either dealation has a slightly higher JH

threshold than vitellogenin absorption by the ovaries, or

dealation proceeds more slowly after the JH threshold is

crossed. Histolysis of the flight muscles, another re-

sponse of virgin queens to queenlessness, takes longer to

occur than does dealation; it begins just after wing

shedding and is complete after about 10 days (Barker,

1979).

In the present study, polygyne virgin queens were

much less responsive than monogyne virgin queens to

removal of the pheromone-producing queens. Keller and

Ross (1993a) also found polygyne queens to be less

responsive. These authors reported that genotype greatly

affects the response of virgin queens from polygyne but

not monogyne colonies. They showed that in polygyne

colonies from Georgia, mature virgin queens with the

genotype Pgm-3"/-3" weighed 13% more than virgin

queens of the other two genotypes that occur at this

locus (Pgm-3"/-3' and Pgm-3b/-3 b, and, following re-

moval of all reproductively active queens, were 5 times

more likely to dealate and begin laying eggs during the

3-day test period. Although the frequency of the Pgm-3

alleles has not been investigated in our Texas study

population, the small number of responsive individuals

608 EDWARD L. VARGO and MICHELE LAUREL

Y

Neural

and/or

chemical 1 0

signal y

CA

3

JH

Low tiler 0

Vitellogenin

FIGURE 4. Proposed general model for the mode of action of the primer pheromone of queen fire ants that inhibits dealation

and ovary development in virgin queens. In this model, the pheromone triggers antenna1 receptors which send inhibitory signals

to the median neurosecretory cells in the brain. Largely inhibited, the median neurosecretory cells only weakly stimulate the

corpora allata to synthesize JH, maintaining low titers of this hormone. At low levels, JH stimulates vitellogenin synthesis in

the fat body. In the absence of the pheromone, the disinhibited neurosecretory cells send a stronger chemical and/or neural

signal that triggers the corpora allata to produce larger quantities of JH. At higher titers, JH stimulates vitellogenin uptake

by the ovaries and dealation, the latter process possibly involving an effect of JH on the nervous system. Dealation may result

from a JH-independent pathway in the nervous system in lieu of or in addition to the JH-mediated pathway. These two possible

pathways for control of dealation are flagged with question marks.

from polygyne colonies found here (12% after 3 days) is

similar to the low frequency of the more responsive

Pgm -3 "l-3" individuals occurring in a Georgia polygyne

population [l 1%; Keller and Ross (1993a)], suggesting

that the frequency of the Pgm-3"/-3" genotype may be

similar in the two populations. Interestingly, this geno-

typic effect on reproductive maturation does not occur

in monogyne colonies, in which all three genotypic

classes are equally likely to undergo reproductive devel-

opment under queenless conditions (Keller and Ross,

1993a).

As hypothesized by Keller and Ross (1993a), the

low responsiveness of virgin queens in polygyne colonies

probably results from the pheromonal milieu of the

colony. Because of the relatively high levels of queen

pheromone that likely accumulate in polygyne colonies

(Vargo and Fletcher, 1987), polygyne virgin queens

are subjected to more intense inhibition than virgin

queens in monogyne colonies. This increased phero-

monal influence likely affects the rate of virgin queen

reproductive maturity in a genotype-specific manner.

Evidence for such a social environment-genotype inter-

action has been recently documented by Keller and Ross

(1993b), in which queen pupae from monogyne and

polygyne colonies were cross-fostered in colonies of the

alternate type. In addition to an effect of social form on

FIRE ANT PRIMER PHEROMONE 609

reproductive development of virgin queens, the results of

the present study indicate there is a strong effect of

source colony in the monogyne form. The causes of this

variability are not known but could involve one or

more of the following: age differences, genotype, social

environment and ecological factors.

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Acknowledgements-We thank W. Donaldson for valuable technical

assistance and L. L. Poulsen for the generous use of his video

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This material is based in part upon work supported by the Texas

Advanced Technology Program under Grant No. 003658134.

... For the expression analysis, the untreated control was used to calibrate the Vg relative expression. As a positive control to validate the application of the JH analog to S. invicta, virgin alate queens were tested following the protocol by Vargo and Laurel (Vargo & Laurel, 1994) since JH application to alate virgin queens causes them to dealate. Twelve hours following the topical application virgin queens were checked for dealation. ...

... The JH analog, S-hydroprene, had no effect on the expression of the four Vgs 12 h after topical application on the abdomen of workers. Previously, it was reported that topical application of a JH analog to virgin queens resulted in queen dealation coupled with some degree of ovary development 8 and 12 h after JH analog application to queens (Vargo & Laurel, 1994). It was suggested that in S. invicta queens, Vg is constitutive, while yolk formation is regulated through the level of Vg uptake into the oocyte, rather than at the level of Vg synthesis by the fat body (Chen et al., 2004). ...

  • Chloe Hawkings
  • Cecilia Tamborindeguy Cecilia Tamborindeguy

Vitellogenin has been proposed to regulate division of labor and social organization in social insects. The red imported fire ant ( Solenopsis invicta ) harbors four distinct, adjacent vitellogenin genes (Vg1, Vg2, Vg3, and Vg4). Contrary to honey bees that have a single Vg ortholog as well as potentially fertile nurses, and to other ant species that lay trophic eggs, S. invicta workers completely lack ovaries or the ability to lay eggs. This provides a unique model to investigate whether Vg duplication in S. invicta was followed by subfunctionalization to acquire non-reproductive functions and whether Vg was co-opted to regulate behavior within the worker caste. To investigate these questions, we compared the expression patterns of S. invicta Vg genes among workers from different morphological subcastes or performing different tasks. RT-qPCRs revealed higher relative expression of Vg1 in major workers compared to both medium and minor workers, and of Vg2 in major workers when compared to minor workers. Relative expression of Vg1 was also higher in carbohydrate foragers when compared to nurses and protein foragers. By contrast, the level of expression of Vg2, Vg3, and Vg4 were not significantly different among the workers performing the specific tasks. Additionally, we analyzed the relationship between the expression of the Vg genes and S-hydroprene, a juvenile hormone analog. No changes in Vg expression were recorded in workers 12 h after application of the analog. Our results suggest that in S. invicta the Vg gene underwent subfunctionalization after duplication to new functions based on the expression bias observed in these data. This may suggest an alternative and still unknown function for Vg in the workers that needs to be investigated further.

... Similar studies have been conducted in social insects. The best example is perhaps in the fire ant Solenopsis invicta, where several studies showed that a putative queen pheromone inhibits wing-shedding and ovary development in virgin queens and is likely produced in multiple exocrine glands including the poison sac, the mandibular glands and possibly other glands (Robert, 1983;VanderMeer and Alonso, 2002;VanderMeer et al., 1980;Vargo, 1999;Vargo and Hulsey, 2000;Vargo and Laurel, 1994). However, the actual pheromone was never identified. ...

  • Etya Amsalem Etya Amsalem

The reproductive division of labour in social insects is a fascinating phenomenon regulated by diverse chemical signals that vary substantially in structure. Is this diversity an example of one problem (reproductive regulation) and many potential solutions (diverse chemicals)? Or are there hidden shared elements in the pheromonal regulation of reproduction across insects? To address this question, I will first discuss the phenomenon of reproductive division of labour in social insects, particularly, the reproductive conflicts among females and the means by which these conflicts are resolved. I will then focus on the use of pheromones, a mode of communication that has broadly diversified among social insects that live in large complex societies. I will summarize the different approaches to define semiochemicals in the context of the reproductive division of labour and review the state of knowledge of compounds regulating insect reproduction, both solitary and social, demonstrating the structural diversity as well as the potential conservation of the mechanisms regulating signal production and perception. Lastly, I will discuss the different hypotheses underlying the evolution of pheromones regulating reproduction in insects. Our current understanding of reproductive signalling, while extensive within single species, is still limited by the paucity of comparative studies across the Insecta as a whole, and further investigations are sorely needed.

... In insects, reproductive dominance is established by behavioural aggression and/or chemical cues (Le Conte and Hefetz, 2008;Padilla et al., 2016). Chemical cues are often in the form of queen pheromones (Matsuura et al., 2010;Van Oystaeyen et al., 2014;Princen et al., 2019;Vargo and Laurel, 1994;Winston and Slessor, 1992). Perhaps the most well-studied queen pheromone is Queen Mandibular Pheromone (QMP), produced by queen honeybees (Apis mellifera) (Keeling et al., 2003;Pankiw et al., 1996;Slessor et al., 1988). ...

Eusociality is characterised by the reproductive division of labour; a dominant female (queen) or females are responsible for the majority of reproduction, and subordinate females are reproductively constrained. Reproductive constraint can be due to behavioural aggression and/or chemical cues, so-called queen pheromones, produced by the dominant females. In the honeybee, Apis mellifera, this repressive queen pheromone is queen mandibular pheromone (QMP). The mechanism by which honeybee workers are susceptible to QMP is not yet completely understood, however it is thought to be through olfaction via the antennae and/or gustation via trophallaxis. We have investigated whether olfaction is key to sensing of QMP, using both Drosophila melanogaster- a tractable non-eusocial insect which is also reproductively repressed by QMP- and the target species, A. mellifera worker honeybees. D. melanogaster are still capable of sensing and responding to QMP without their antenna and maxillary palps, and therefore without olfactory receptors. When worker honeybees were exposed to QMP but unable to physically interact with it, therefore required to use olfaction, they were similarly not reproductively repressed. Combined, these findings support either a non-olfactory based mechanism for the repression of reproduction via QMP, or redundancy via non-olfactory mechanisms in both D. melanogaster and A. mellifera. This study furthers our understanding of how species are susceptible to QMP, and provides insight into the mechanisms governing QMP responsiveness in these diverse species.

... It influences physiology in queens and guarding behavior in workers of primitive eusocial wasp Polistes canadensis (Giray et al., 2005). JH also affects queen maturation and reproduction in the invasive ant S. invicta where, high levels of JH induces alates to begin oogenesis (Vargo and Laurel, 1994;Brent and Vargo, 2003;Lu et al., 2009). Vg is a yolk precursor protein; its production is typically used to produce egg yolk by oviparous animals (Amdam et al., 2003), but it may also affect behavior (Nelson et al., 2007). ...

Many species of social Hymenoptera demonstrate behavioral flexibility, where older workers that typically forage can revert to younger worker tasks, such as nursing, when these are absent. This flexibility is typical of the sterile worker class, yet rare in queens. In the little fire ant (Wasmannia auropunctata), queens have been reported to perform only egg laying. We examined behavior of queens of W. auropunctata after demographic manipulation. When half of the workers were removed from the colony, queens were observed caring for eggs, larvae and pupae as well as eating outside of the nest, like forager workers. We examined the relationship between these atypical queen behaviors and their juvenile hormone binding protein (JHbp) and vitellogenin (Vg) expression via QRT-PCR method. JHbp and Vg expression decreased when queens were performing worker tasks, resembling the expected expression pattern of typical sterile workers. Flexibility in queen behaviors in the little fire ant may be an important adaptation to changing environments. As a significant invasive species, such adaptation may increase the probability of colony survival during propagation. Our results not only present new insights in behavioral flexibility in social insects, but also increases our understanding of the success of this significant invasive species.

... To 48 achieve this in the Hymenoptera, a clade containing many eusocial species, a mixture of 49 behavioural aggression and chemical inhibition of reproduction is used (Le Conte and Hefetz, 50 2008; Padilla et al., 2016). Chemical inhibition occurs via queen pheromones (Matsuura et 51 al., 2010;Vargo and Laurel, 1994; Winston and Slessor, 1992). These queen pheromones are 52 produced by the reproductively dominant female and are thought to signal her fecundity to 53 ...

Queen pheromones effect the reproductive division of labour, a defining feature of eusociality. Reproductive division of labour ensures that one, or a small number of, females are responsible for the majority of reproduction within a colony. Much work on the evolution and function of these pheromones has focussed on Queen Mandibular Pheromone (QMP) which is produced by the Western or European honeybee (Apis mellifera). QMP has phylogenetically broad effects, repressing reproduction in a variety of arthropods, including those distantly related to the honeybee such as the fruit fly Drosophila melanogaster. QMP is highly derived and has little chemical similarity to the majority of hymenopteran queen pheromones which are derived from cuticular hydrocarbons. This raises the question of whether the phylogenetically widespread repression of reproduction by QMP also occurs with more basal saturated hydrocarbon-based queen-pheromones. Using D. melanogaster we show that saturated hydrocarbons, are incapable of repressing reproduction, unlike QMP. We also show no interaction between the four saturated hydrocarbons tested or between the saturated hydrocarbons and QMP, implying that there is no conservation in the mechanism of detection or action between these compounds. We propose that the phylogenetically broad reproductive repression seen in response to QMP is not a feature of all queen pheromones, but unique to QMP itself, which has implications for our understanding of how queen pheromones act and evolve.

... Queen signals are a subset of caste-recognition signals that distinguish the queen from the workers [5 ]. Within Hymenoptera, such pheromones were only found in A. mellifera where the composition of the pheromone is known [18 ], and in Solenopsis invicta where the composition remains elusive [19,20]. The A. mellifera queen mandibular pheromone blend is responsible for maintenance of the queen reproductive monopoly by inducing worker sterility [21,22]. ...

Pheromones mediating social behavior are critical components in the cohesion and function of the colony and are instrumental in the evolution of eusocial insect species. However, different aspects of colony function, such as reproductive division of labor and colony maintenance (e.g. foraging, brood care, and defense), pose different challenges for the optimal function of pheromones. While reproductive communication is shaped by forces of conflict and competition, colony maintenance calls for enhanced cooperation and self-organization. Mechanisms that ensure efficacy, adaptivity and evolutionary stability of signals such as structure-to-function suitability, honesty and context are important to all chemical signals but vary to different degrees between pheromones regulating reproductive division of labor and colony maintenance. In this review, we will discuss these differences along with the mechanisms that have evolved to ensure pheromone adaptivity in reproductive and non-reproductive context.

... However, several lines of evidence suggest that JH and biogenic amines play a central role in QP-mediated effects. First, the effects of QPs on worker ovarian development and larval caste can be mimicked by topical application of methoprene, a JH analogue (Edwards 1987;Penick and Liebig 2012;Vargo 1992;Vargo and Laurel 1994). In a social wasp, a recent study has shown that this treatment also affects CHC synthesis (Oliveira et al. 2017). ...

Ant queen pheromones (QPs) have long been known to affect colony functioning. In many species, QPs affect important reproductive functions such as diploid larvae sexualization and egg-laying by workers, unmated queens (gynes), or other queens. Until the 1990s, these effects were generally viewed to be the result of queen manipulation through the use of coercive or dishonest signals. However, in their seminal 1993 paper, Keller and Nonacs challenged this idea, suggesting that QPs had evolved as honest signals that informed workers and other colony members of the queen's presence and reproductive state. This paper has greatly influenced the study of ant QPs and inspired numerous attempts to identify fertility-related compounds and test their physiological and behavioral effects. In the present article, we review the literature on ant QPs in various contexts and pay special attention to the role of cuticular hydrocarbons (CHCs). Although the controversy generated by Keller and Nonacs' (Anim Behav 45:787–794, 1993) paper is currently less intensively debated, there is still no clear evidence which allows the rejection of the queen control hypothesis in favor of the queen signal hypothesis. We argue that important questions remain regarding the mode of action of QPs, and their targets which may help understanding their evolution.

... Yet, queen pheromones still exist, for instance in fire ants; here they have other social functions such as inhibiting the development of virgin queens within the natal nest (e.g. Vargo and Laurel 1994). ...

  • Judith Korb Judith Korb

Termites evolved eusociality independently from social Hymenoptera. As a common trait, reproductive monopoly is maintained through chemical communication. The queen (and in termites also a king) prevents workers from reproduction by conveying their reproductive status. In termites all soldiers are sterile, but workers' potential to reproduce differs between species. It ranges from totipotency in wood-dwelling lower termites where workers are a transient stage from which all other castes develop, to sterile workers in some higher termites. Intermediate are species in which workers can develop into replacement sexuals within the nest but not into winged sexuals. I summarize the patchy picture about fertility signaling that we currently have for termites, pointing also to potential conflicts over reproduction that differ from those in social Hymenoptera. Recent findings imply that, similar to many social Hymenoptera, wood-dwelling termites that live in confined nests use long-chain cuticular hydrocarbons (CHCs) as fertility signals. Yet other compounds are important as well, comprising proteinaceous secretions and especially volatiles. For a subterranean termite, two volatiles have been identified as primer pheromones that prevent reproductive differentiation of workers. It requires more data to test whether wood-dwelling termites use CHCs, while species with larger colonies and less confined nests use volatiles, or whether all species rely on multicomponent signals. Ultimately, we need more effort to model and test potential conflicts over reproduction between queens, kings and workers. Here results from social Hymenoptera cannot be transferred to termites as the latter are diploid and commonly inbred. This review illustrates promising future research avenues.

  • Haolin Zeng
  • Jocelyn G. Millar
  • Li Chen
  • Kenneth G. Ross

Ants use chemical signals to communicate for various purposes related to colony function. Social organization in the red imported fire ant, Solenopsis invicta, is determined by the Sb supergene, with colonies of the monogyne (single-queen) form lacking the element and colonies of the polygyne (multiple-queen) form possessing it. Polygyne workers accept new reproductive queens in their nest, but only those carrying Sb; young winged queens lacking this genetic element are executed as they mature sexually in their natal nest or as they attempt to enter a foreign nest to initiate reproduction after mating and shedding their wings. It has been suggested that queen supergene genotype status is signaled to workers by unsaturated cuticular hydrocarbons, while queen reproductive status is signaled by piperidines (venom alkaloids). We used high-throughput behavioral assays to study worker acceptance of paper dummies dosed with fractions of extracts of polygyne queens, or blends of synthetic counterparts of queen cuticular compounds. We show that the queen supergene pheromone comprises a blend of monoene and diene unsaturated hydrocarbons. Our assays also reveal that unsaturated hydrocarbons elicit discrimination by polygyne workers only when associated with additional compounds that signal queen fertility. This synergistic effect was obtained with a polar fraction of queen extracts, but not by the piperidine alkaloids, suggesting that the chemical(s) indicating queen reproductive status are compounds more polar than cuticular hydrocarbons but are not the piperidine alkaloids. Our results advance understanding of the role of chemical signaling that is central to the regulation of social organization in an important invasive pest and model ant species.

Reproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour's gland secretion in the primitively eusocial bumble bee Bombus impatiens signals information about caste, social condition, and reproductive status. We chemically analyzed Dufour's gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts. We found that workers and queens each possess caste-specific compounds in their Dufour's glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour's gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour's gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.

  • C.G. Butler
  • E.M. Fairey

1.Ovary development in worker honeybees is inhibited by 9-oxodecenoic acid or its vapour. A scent from the queen, in addition to her 9-oxodecenoic acid, also inhibits oogenesis in workers, but it is less effective than the acid. The scent and acid do not work together synergically. Neither 9-oxodecenoic acid nor queen scent, nor the two together, are as effective in inhibiting oogenesis in workers as access to a live queen. Older, mated queens are more effective than younger, virgin ones. Injection of 9-oxodecenoic acid partially inhibits ovary development in workers, but does not inhibit queen rearing by them. The mode of action of this acid is still uncertain.

  • D. P. Jouvenaz
  • G. E. Allen
  • W. A. Banks
  • Daniel P. Wojcik

In a survey conducted in the Southeastern United States, one colony in a sample of 1,007 colonies of the red imported fire ant, Solenopsis invicta Buren, was infected with a microsporidium (Protozoa: Microsporida). The normal host of this parasite appears to be the tropical fire ant, Solenopsis geminata (F.). A benign or very mildly pathogenic yeast was associated with 93 (9.24%) of the S. invicta colonies, and was most common in areas which have been infested with this ant for the longest periods. No pathogens were associated with 83 colonies of the black imported fire ant, Solenopsis richteri Forel. The apparent rarity of bona fide pathogens in imported fire ants in the United States is in marked contrast to the abundance of pathogens in these and other Solenopsis spp. in South America. Four species of microsporidia (possibly new genera) were detected in 22 (7.2%), 12 (3.9%), 6 (2.0%), and 4 (1.3%) of 307 colonies of the tropical fire ant, S. geminata. One colony of this species was infected by a neogregarine (Sporozoa: Neogregarinida). No pathogens were found in a small sample (53 colonies) of the Southern fire ant, Solenopsis xyloni McCook.

  • Mark L. Winston
  • Keith N. Slessor

Describes new findings on the mechanisms of coordination of individuals in an Apis mellifera colony. Discusses the composition and role of pheromones on behaviour and development of the colony rearing new queens and the development of ovaries in worker bees; how the fractions of the queen's pheromones that control the retinue response were identified; the functions of the queen's pheromones, including the presence/absence effects on the number of new queens raised and the control of swarming. The quantities of pheromones produced by the queen and how these were transmitted through the colony by retinue workers were studied, showing that most of the pheromones are internalised. -L.J.Chellis