to b or not to b: a pheromone-binding protein regulates colony social organization in fire ants

To b or not to b:a pheromone-bindingprotein regulates colonysocial organization in fire antsMichael J.B. Krieger

SummaryA major distinction in the social organization of antsocieties is the number of reproductive queens thatreside in a single colony. The fire ant Solenopsis invictaexists in two distinct social forms, one with coloniesheaded by a single reproductive queen and the othercontaining several to hundreds of egg-laying queens.Thisvariation insocial organizationhasbeenshown tobeassociated with genotypes at the geneGp-9. Specifically,single-queen colonies have only the B allelic variant ofthis gene, whereas multiple-queen colonies always havethe b variant as well. Subsequent studies revealed thatGp-9 shares the highest sequence similarity with genesencoding pheromone-binding proteins (PBPs). In otherinsects, PBPs serve as central molecular componentsin the process of chemical recognition of conspecifics.Fire ant workers regulate the number of egg-layingqueens in a colony by accepting queens that produceappropriate chemical signals and destroying those thatdonot. The likely role ofGP-9 in chemoreception suggeststhat the essential distinction in colony queen numberbetween the single and multiple-queen form originatesfrom differences in workers’ abilities to recognizequeens. Other, closely related fire ant species seem toregulate colony social organization in a similar fashion.BioEssays 27:91–99, 2005.� 2004Wiley Periodicals, Inc.

Introduction

It is rather challenging to write an article under the heading of

‘‘my favorite molecule’’ for the reason that an enormous

amount of knowledge has accumulated over the last two

decades pertaining to the molecule’s invertebrate host, but

much less so with respect to the molecule itself. The host in

question is the invasive pest species Solenopsis invicta, the

red imported fire ant.

In 1997, Ken Ross discovered that colony social organi-

zation of S. invicta was associated with different genotypes

at the codominant protein locus general protein-9 (Gp-9 ).(1)

At the time, it was not known whether the gene product of

Gp-9 was directly involved in determining social organization

nor was it known to what protein family it may belong. Having

worked on the evolution of social systems myself,(2–4) I was

fascinated by the possibility that a few genes, perhaps even a

single gene could shape social organization in an ant species.

In order to address this fascinating subject matter, I joined the

Ken Ross laboratory to characterize the underlying genetic

architecture of this protein, and if possible to understand its

influence on social organization.

To fully appreciate the findings on this interesting protein as

well as the genetic basis of social organization in fire ants, it is

first necessary to understand the basic features of its natural

history and social biology. I will first describe the relevant

features of the biology of S. invicta, detailing the major dif-

ferences that characterize the two social forms of this species.

Then I will continuewith a summary of the genetic data showing

that the geneGp-9 is amajor candidategene influencing social

organization in S. invicta and some other fire ant species.

Background biology of S. invictaS. invicta, a South American native, was accidentally intro-

duced to the US, probably around 1920 to the port of Mobile,

Alabama.(5) Since its introduction, this ant has established

itself throughout the southeastern US and more recently in

California. Due to its high population densities, painful sting

and negative impact on native wildlife and agriculture,

S. invicta is considered a major economic and ecological pest

in the US.

Amajor distinction in the social organization of ant societies

is the number of queens that co-exist in a colony.(6) Some

species or populations have colonies that always contain a

single queen whereas others have colonies that contain

multiple queens. InS. invicta, both social forms exist and even

occur at times in the same habitat. Colonies of the single-

queen form (monogyne form) are simple families headed by a

single reproductive queen, whereas colonies of the multiple-

queen form (polygyne form) contain several to hundreds

of egg-laying queens.(7,8) The two social forms also differ in

other important traits besides number of queens per nest,(9)

including dispersal strategies, mode of colony founding and

energy reserves of young queens.

BioEssays 27:91–99, � 2004 Wiley Periodicals, Inc. BioEssays 27.1 91

Center for Studies in Physics and Biology, Rockefeller University,

New York, NY 10021-6399, USA. E-mail: [email protected]

DOI 10.1002/bies.20129

Published online in Wiley InterScience (www.interscience.wiley.com).

My favorite molecule

Young monogyne queens store large quantities of energy

reserves in the formof fat andglycogen,which enables them to

disperse over a relatively long distance.(10) A more important

effect of these large energy reserves is however, the queen’s

ability to found new colonies without the assistance of workers

(independently). After having mated with a single male in mid-

air, monogyne queens land, dig a chamber in the soil and start

laying eggs. Since queens do not feed during the initial stages

of colony founding, they rear their first clutch of workers

entirely on their own energy reserves.(11,12) In contrast, most

polygyne queens do not accumulate sufficient energy reserves

during their maturation to found new nests independently.

Instead, they seek adoption into already existing polygyne

nests where they initiate reproduction with the help of the

existing worker force.

General protein-9 (GP-9)

The fact that colony social form was associated with different

Gp-9 genotypes was quite surprising because the two forms

strongly resemble one another genetically otherwise, typically

harboring the same alleles and level of genetic variation at

numerous genetic markers.(13,14) The genotypic pattern asso-

ciated with each social form is remarkably simple: monogyne

colonies harbor only the B allelic variant of Gp-9, whereas

polygyne colonies always have the b variant as well. Accord-

ingly, monogyne queens are always BB homozygous, and

mate with a single, haploid male also bearing the B allele,

resulting in female offspring that all bear theBB genotype.(1,15)

In contrast, reproductive queens in polygyne colonies are

always Bb heterozygotes but produce offspring with all three

genotypes (BB, Bb, bb). However, only queens with the Bb

genotype will become egg layers in polygyne colonies. This

puzzling pattern arises through a complex interaction of queen

phenotype, queen–worker interaction and mode of colony

founding (Fig. 1).

Thegenotype atGp-9 is strongly associatedwith theweight

of young queens due to differential accumulation of fat during

adult maturation.(16–18) Young queens with the homozygous

BB genotype gain themost weight, regardless of whether they

were raised in a monogyne or polygyne colony. Heterozygous

queens gain intermediate weight and the bb homozygotes

gain no weight at all (Fig. 2). The molecular basis for the

differential weight gain is currently unknown. Perhaps GP-9

exerts this effect indirectly, perhaps currently unidentified,

tightly linked genes are responsible for this effect.

Asmentioned above, the amount of energy reserves stored

by queens determines their reproductive strategy. Relatively

extensive reserves are necessary to carry a queen through

independent colony founding,whichcanonly beaccomplished

by queens with the BB genotype. In contrast, joining estab-

lished polygyne colonies does not require extensive energy

reserves and is easily accomplished by the lighter Bb queens

(most bb queens die before they reach sexual maturity).

Theworkers in a fire ant colony resolvewhich young queens

will be accepted as new egg layers, which will be rejected by

execution and how many queens are tolerated as permanent

reproductives. Their decision is believed to depend on the

presence or absence of specific queen pheromones.(19)

In monogyne colonies, no additional reproductive queen of

any genotype is ever tolerated besides the mother

queen.(16,17,20,21) All intruder queens are seized and executed

upon entering the colony, controlled by the queen’s pheromo-

nal signals. Identification of intruder queens might be further

enhanced by the presence of colony-specific recognition

cues derived from heritable and environmental (food, soil)

sources(22) that are implicated in the highly aggressive

behavior of monogyne workers towards non-nestmates.(23)

In contrast, polygyne colonies display little or no intercolony

discrimination.(23) Despite the tolerance towards non-colony

members, only Bb queens are accepted in multiples as new

reproductives into polygyne colonies, all BB queens are

executed.(16,17,20,21)

In short, monogyne colonies are initiated by a single queen

carrying the BB genotype. All offspring carry the same BB

genotype as their mother, providing the sexual offspring with

extensive energy reserves that enables them to found new

monogyne nests. Carrying this genotype however, makes it

impossible to become reproductively active in any polygyne

colony, as any attempt will lead inevitably to their execution by

polygyne workers. Polygyne colonies contain only hetero-

zygous reproductives but produce young queens of all three

genotypes. However, the bb homozygotes die before sexual

maturity, most of the BB queens approaching sexual maturity

are executed and only the Bb heterozygotes mature in

good numbers to become egg layers themselves. Most

importantly however, only queens carrying the Bb genotype

are ever accepted as new reproductives in polygyne colonies.

Conversely, the lower energy reserve of this genotype

prevents the Bb queens from founding nests independently

(Fig. 1).

What protein is encoded by Gp-9?Gp-9 was regularly used in our laboratory as one of many

protein markers to assess population genetic structure in fire

ant populations.(13,14) Each marker represents a protein that

exists in distinct variants, differing in their electrophoretic

mobility. These protein variants are first separated on starch

gels by their charge differences and then stained in order to

visualize the banding pattern. For known, enzymatic proteins,

the protein stain typically contains the substrate and all the

cofactors requiredby theenzyme to catalyze its reaction. In the

case of non-enzymatic or unknown proteins, the visualization

of the banding phenotypes is accomplished using nonspecific

protein staining. GP-9 was in the latter category of unknown

proteins, as might be hinted from its non-predicative name

(general protein-9).


92 BioEssays 27.1

We determined the amino acid sequences of some of

the peptide fragments isolated from the starch gel and

subsequently used this information to recover the full-length

mRNA transcript of Gp-9.(24) Determination of the nucleotide

sequence of Gp-9 and the predicted amino acid sequence

of its protein product revealed that it shares the highest

sequence similarity with genes encoding pheromone-

binding proteins (PBP), a subclass of the odorant-binding

protein (OBP) family.(25) OBPs are mainly expressed in the

antenna and are characterized by six absolutely conserved

cysteine residues located in similar positions.(26,27) They

transport odorants from the porous sensillum wall to the

receptors located on the dendritic membrane of the olfactory

sensory neurons.(27) These neurons respond to a range

of odors such as plant volatiles, food sources,(28) and

conspecifics.(29)

Figure 1. Colony cycle of the monogyne and polygyne social form. MonogyneBB homozygous queens mate with a single, haploid male

also bearing the B allele, resulting in female offspring that all bear the BB genotype. After the mating flight, monogyne queens found new

nests independently, and rear their first brood entirely on accumulated fat reserves. The BB genotype provides young queens with large

energy reserves needed to found new monogyne nests. Carrying the BB genotype, however, makes it impossible to be accepted into

polygyne colonies, as any attempt will lead to their inevitable execution. Polygyne colonies contain only Bb queens but produce young

queens of all three genotypes (BB, Bb, bb). Yet, only the Bb queens survive to maturity and become egg layers by seeking adoption into

existing polygyne colonies. Bb queens do not accumulate enough fat reserves to initiate new nests independently (see text for details).


BioEssays 27.1 93

Fire ant workers regulate the number and identity of

egg-laying queens in a colony by accepting queens that

produce appropriate chemical signals and destroying those

that do not.(19,21,30) Thus, the core feature of colony social

organization, the number of egg-laying queens, is mediated

by worker recognition and subsequent discrimination among

queens. The presumed role of GP-9 in chemoreception

suggests that the essential distinction in colony queen number

between the monogyne and polygyne forms is strongly

connected to differences in workers’ abilities to recognize

queens.

The Gp-9 alleles in S. invictaOur first undertaking after the identification of Gp-9 as a

PBP gene was to link the protein variation to the underlying

nucleotide variation. Accordingly, we sequenced Gp-9 alleles

from numerous individuals of both social forms collected

throughout the introduced range in the US. We found two

variants, and these corresponded in every case to the two

alleles identified by protein electrophoresis (B and b).

Remarkably, the two Gp-9 alleles differed by nine nucleotide

substitutions in their coding regions, each of which is asso-

ciated with an amino acid substitution.(24) This high ratio of

nonsynonymous to synonymous substitutions suggests that

positive selection has driven the divergence of these

alleles,(31) consistent with behavioral studies implicating

strong diversifying selection on Gp-9.(1,32) The correspon-

dence of the sequence variants and electrophoretically deter-

mined alleles was further confirmed by identifying the charge-

changing amino acid substitution at position 151, responsible

for the different electrophoretic mobilities of the allelic proteins

in starch gels. Thus, the allelic pattern that emerged from the

nucleotide data corresponded unerringly with the pattern seen

at theprotein level: theBallele is retrieved frommonogyneand

polygynepopulations,whereas theballele is foundexclusively

in polygyne populations.

This pattern led to thehypothesis that theballele is required

for the expression of the polygyne social form. However,

previous protein electrophoretic studies of S. invicta from the

native range in Argentina showed that, although the b allele

is found only in the polygyne form, some nests of this form

contain egg-laying queens scored electrophoretically as BB

homozygotes. These findings, if confirmed at the nucleotide

level, would have nullified our hypothesis that the b allele is

required for the expression of the polygyne social form. By

sequencing appropriate samples from the native range, we

found that these polygyne queens were also heterozygotes

at Gp-9, but possessed a ‘‘cryptic’’, functionally b-like allele

(b 0) that encodes a protein bearing the net charge of, and

thus electrophoretically indistinguishable from, a B allele

product. This ‘‘cryptic’’ b 0 allele is more similar to the b than

theB allele over its entire coding sequence yet bears the same

charge-conferring amino acid as the B allele at position 151

(Fig. 3A).

Figure 2. Weight gain of maturing queens of different Gp-9

genotypes and social organization. Mean weights are pre-

sented with their corresponding standard deviations.(16,18,20)

Figure 3. Amino acid variation for the alleles of Gp-9. A: Socially polymorphic species from the ‘‘South American’’ fire ant clade. The

alleles separate into two distinct groups, the B-like and the polygyny-permitting b-like alleles. Amino acid substitutions uniquely shared

amongallb-like alleles, are indicatedby circles, the charge-changing aminoacid substitution in theb allele ofS. invicta is indicated by a star.

B: Amino acid composition at the same positions in S. geminata, a species belonging to the ‘‘North American’’ fire ant clade.(24,25)


94 BioEssays 27.1

This confirmed link of allelic pattern and social form in

the native range of S. invicta led to the possibility that

the expression of polygyny in other species might be similarly

associated with the genotypic pattern atGp-9. To this end, we

sequencedGp-9 in nineotherSolenopsis species, aswell as in

a species of a related genus, to establish the taxonomic range

overwhich a homolog occurs.Wewere unable to amplifyGp-9

in the related genus, suggesting that if such a homolog exists,

it must have undergone extensive sequence divergence at the

primer-binding sites.

Gp-9 alleles in other fire ant species

We used sequence data from the ten Solenopsis species to

reconstruct the evolutionary relationships of theGp-9 variants

(Fig. 4). The evolutionary relationships were found to be con-

sistent with the classification of these ants,(33,34) forming

two large groups—the ‘‘North American’’—and the ‘‘South

American’’ fire ant clade.(24) Most interestingly, Gp-9 se-

quences from the species in the South American clade known

to display polymorphism in social organization are further

divided in sister clades, one containing the close relatives of

the polygyny-permitting b allele of S. invicta and the other

containing the close relatives of the B allele of S. invicta. As

expected if alleles in the b-like clade induce polygyny, queens

from confirmed polygyne nests of three species (in addition to

S. invicta) invariably carried such b-like alleles. The deduced

phylogeny allowed us to infer that the ancestral Gp-9 allele

for the socially polymorphic clade was of the B type, and,

hence, that monogyne social organization preceded polygyny

in the evolutionary history of South American fire ants. The two

other South American species that are most closely related to

the socially polymorphic species, are not known to exhibit

polygyny. Their lack of a b-like allele is consistent with the

hypothesis that b-like alleles are necessary for the expression

of polygyny (Fig. 4).

The implied single origin of b-like alleles in these ants

apparently predated the origins of most of the species, sug-

gesting that the expression of polygyny in each was made

possible by survival of the descendants of an ancestral b-like

allele through sequential speciation events.

The availability of a gene phylogeny for Gp-9 also made it

possible to investigate the role of selection in the evolutionary

history of this gene. We tested the specific hypothesis that the

b-like alleles, which are integral to the polygyne social system,

are under different selective regimes than the B-like alleles,

which must function in both social systems. As hypothesized,

positive selection was statistically significant only on branches

within the b-like clade. Despite positive selection having acted

periodically on various b-like alleles to drive their divergence

from their B-like counterparts, all b-like alleles uniquely share

the three amino acids G42, I95 and I139 (Fig. 3A), suggesting

that one or more of these residues are essential for the

expression of polygyny. It is therefore of particular interest to

investigate their relative position on the protein structure,

perhaps allowing us to infer the molecular mechanism by

which polygyny is induced. Unfortunately, we do not have the

structure of GP-9 at our disposal, but computerized sequence

alignment algorithms in combination with threading techni-

ques(35,36) allows us to arrive at reasonably accurate structure

prediction, assuming appropriate structural templates are

supplied.

Structural hypothesis

Currently there are four OBP three-dimensional structures

available on the Protein Data Bank: a silkmoth PBP,(37–39) a

cockroach PBP,(40) a fruit fly OBP(41) and, most recently, a

honey bee PBP.(42) While all these OBPs display similar folds,

their binding pockets comprise a variety of shapes and binding

affinities suggesting that the OBP fold is fairly versatile and

able to bind a considerable range of organic compounds.

Figure 4. Cladogram illustrating the phyloge-

netic relationship of the Gp-9 alleles from ten

Solenopsis species. The species separate into two

groups, the ‘‘North American’’ and the ‘‘South

American’’ fire ant clade.Gp-9 sequences from the

South American clade of fire ant species known

to display polymorphism in social organization

form sister clades, one containing the polygyny-

permitting b-like alleles, the other containing the

B alleles. The tree is rooted using the sequence

from thenon-fire-ant speciesS. globularia littoralis.


BioEssays 27.1 95

The silkmoth PBP undergoes a pH-dependent conforma-

tional change that results in a structure that is unable to bind

the ligand. Specifically, the C-terminal tail of the protein forms

an a-helix that folds into the binding pocket at low pH, blocking

the ligand-binding site.(38) This has led to the hypothesis that

ligands are unloaded through a conformational change of their

carrier protein as they approach the acidic membrane.(39)

However, pH-dependent conformational change does not

seem to be an universal feature involved in unloading ligands

in all OBPs, as only the silkmoth protein has such an extended

C terminus that folds inside the protein at acidic pH. The other

threeOBPproteins either lack this C-terminal tail entirely(40) or

carry only a short extended irregular structure, which, rather

than formingamobile helix, is part of the cavitywall. This stable

structural configuration is unaffected at different physiological

pH values.(41,42)

To establish the significance of eachof the three aminoacid

residues consistently associated with the polygyny-permitting

b-like alleles, wemapped these residues to theGP-9 structure

prediction obtained by GenTHREADER(35) and the Robetta

server.(36) We found that two of the residues, Ile95 and Ile,139

are part of the cavity wall that surrounds the binding pocket.

Residue Ile139 even extends its side chain into the binding

pocket, an indication that Ile139 might be directly involved in

ligand binding. The third residue, Gly42 is located on a solvent

exposed loop-like structure that is not part of the cavity wall.

The change fromaserine to a glycine on this loop probably has

little effect on the protein structure as both amino acids are

similar in size and exhibit similar hydrophobicities. However,

the two substitutions located on the cavity wall (M95I, V139I),

although not radically different, are more likely to have an

effect on the binding of the ligand.

The initial discovery of Gp-9 and its association with social

form in S. invicta was due to the differential electrophoretic

mobility of the two protein alleles, caused by a single charge-

changing amino acid substitution at position 151 in the b allele.

A second, ‘‘cryptic’’ b 0 allele was discovered in Argentina that

encodes another functional b-like protein but bears the net

charge of the B allele. The three additional South American

polygyne species also invariably possess b-like alleles that

lack the charge-changing amino acid substitution. Thus,

only one allele, the b allele of S. invicta harbors the charge-

changing amino acid substitution (Fig. 3A). This substitution

maps to the C-terminal tail of the silkmoth PBP, the structure

that is supposedly responsible for unloading the ligand or,

in the case of the fruit fly or honey bee protein, to the irregular

C-terminal structure that is a part of the cavity wall. The C-

terminal tail ofGP-9 is slightly shorter than theC-terminal tail of

the silkmoth protein, yet longer than the fruit fly or honey bee

protein. Hence, it is difficult to assess with certainty which of

the two structures GP-9 more closely resembles at its C-

terminal tail. However, in both cases, it is likely that a change

from a basic lysine to an acidic glutamic acid will cause the

PBP to lose its ability to bind or to release the ligand. A second

possibility, as parts of the C-terminal tail of the silkmoth PBP

are also involved in dimer formation,(37) is that the charge-

changing substitution prevents proper dimer formation of

GP-9 and, hence, renders the molecule biologically non-

functional.

Interestingly, these structural hypotheses implicating the

lack of function of the b allele protein, is consistent with the

differential weight gains of young maturing queens according

to theirGp-9 genotypes (Fig. 2). According to these structural

hypotheses, bb homozygotes do not express any functional

GP-9 protein, consistent with the lack of weight gain during

their maturation process. Heterozygous queens will generate

50%of their total GP-9 production in a functional formand they

gain intermediate weight, and finally, the BB queens produce

only functional GP-9 proteins and hence gain themost weight.

While the details of such a direct relationship between weight

gain and protein structure remain difficult to understand, it

reveals that the charge-changing amino acid substitution

occurring in the b allele ofS. invicta and the three substitutions

commonly shared among all b-like alleles have two distinct

effects. According to this hypothesis, polygyny is induced by at

least one of the three amino acid substitutions shared among

all b-like alleles whereas the differential weight gains of young

S. invicta queens and the associated low viability of the bb

homozygotes is caused by the charge-changing amino acid

substitution. This leads to a testable prediction, namely that

polygyne colonies harboring b-like alleles that lack the charge-

changing amino acid substitution should contain viable

reproductive queens with a bb-like genotype. Preliminary data

from a recent population screen in S. richteri from South

America suggest that this hypothesis is correct (unpublished

data).

Are the molecular mechanisms underlying

social organization in fire ants universal?

Allelic determination of polygyny was found in four closely

related species in the South American fire ant clade. The

ancestralGp-9 allele for the socially polymorphic clade was of

the B type, implying that the monogyne social organization

precededpolygyny in theSouthAmerican fire ants and that the

b-like alleles originatedwithin that clade.Outside of this group,

polygyny is unfortunately only well documented in a single

North American species, S. geminata(43,44) Nevertheless, the

occurrence of polygyny in S. geminata, makes it possible to

address the question of whether allelic determination of social

organization represents an universal mechanism that can be

found in other ant species as well. We had already sequenced

the Gp-9 sequence of a monogyne S. geminata specimen in

our initial study(24) and this sequence features the char-

acteristic B-like residues methionine and valine at positions

95 and 139, respectively (Fig. 3B). However, the sequence

includes the b-like glycine residue at position 42, suggesting


96 BioEssays 27.1

that the amino acid at this position may not be essential to the

function of GP-9 protein with respect to social organization.

Interestingly, this is the same residue that was found on the

loop-like structure of the GP-9 protein predicted by the protein

structure prediction software.

In our search for polygyneS. geminata specimens, Sanford

Porter, a fire-ant researcher from Gainesville Florida, directed

us to small, isolated polygyne population consisting of

approximately twenty-five nests, nestled within a small strip

of land among a collection of otherwise monogyne nests.

Despite extensive search efforts, wewere unable to locate any

other polygyne population in theUS. The sequence analysis of

the polygyne Gp-9 sequences revealed that the amino acid

replacements characteristic of all b-like alleles were not

present in polygyne S. geminata(45) disproving our original

hypothesis that one or both of these substitutions are

necessary for the expression of polygyny in all fire ants. We

found, however, that the polygyne form lost a great deal of

genetic diversity at both their nuclear and mitochondrial

genomes relative to the monogyne form. This led us to

speculate that the polygyne form of S. geminata originated

from a small, isolated founder population derived from the

nearby monogyne population. In this view, the origin of

polygyny in S. geminata was driven by a loss of genetic

variation rather than by specific amino acid replacements at

Gp-9, the evolutionary event that drove the origin of polygyny

in the South American fire ant clade species.

Loss of genetic variation has been previously invoked to

account for shifts in colony social organization. For example, in

the Argentine ant (Linepithema humile), a change in the ability

to recognize nestmates, a feature well developed in its native

South American range, but lost in the introduced ranges, has

led to the formation of massive ‘‘supercolonies’’ in which

individual ants mix freely among physically separated

nests.(46–48) Reduced nestmate recognition in the introduced

ranges coincides with loss of genetic diversity. These obser-

vations have led to the idea that loss of alleles encoding

chemical recognition cues, caused by the founder events,(48,49)

have eroded the nestmate discrimination abilities of Argentine

ant workers in their introduced ranges, thereby inducing a shift

in colony social organization.

Future directions

Our research on the molecular mechanism of social organiza-

tion in fire ants revealed two possible routes leading to the

polygyne social form. Both scenarios invoke changes in

the molecular components of the chemoreception systems,

although in a different manner. In the South American fire

ant clade, polygyny evolved presumably by a change in the

binding affinity of GP-9, altering recognition capabilities of

workers that bear b-like alleles. Evolution of polygyny in S.

geminata via loss of alleles at loci encoding recognition cues

seems to involve a reduction in the diversity of chemical

labels necessary for the proper functioning of a discrimination

system that serves in regulating queen number.

Several issues regarding Gp-9 and its role in determining

social organization are still unresolved and need further

attention. First, what is the molecular basis for the differential

weight gain according to the Gp-9 genotypes in young

S. invicta queens? Is GP-9 exerting this effect indirectly,

caused by a change in the binding affinity of the b allele? For

example, a change in the binding affinity could altered odor

perception thereby causing queens to behave atypically.

Since it is the workers that feed young queens, unchar-

acteristic queen behavior may lead to a neglect by workers,

resulting in lower body weights of queens carrying the b allele.

Alternatively, the differential weight gain is not a result of the

different Gp-9 alleles but brought about by currently unidenti-

fied genes, tightly linked to Gp-9. To explore this idea, we

intend to sequence an approximately 200-kb region around

the B and b allele of the Gp-9 gene in order to find candidate

genes that might affect weight gain. Differences in the DNA

sequences of genes associatedwith the alternateGp-9 alleles

will point to such candidate genes.

A second unresolved issue is the acceptance of only Bb

queens into polygyne colonies. Why are all BB queens ex-

ecuted but most of the Bb queens entering polygyne colonies

are left unharmed? The reason for this phenomenon is not

known, but we speculate it is a combination of three factors.

Two of the factors reduce worker aggression in general, and

one relates specifically to Bb queens. The most important

factor, we believe, is the presence of Bb workers in polygyne

colonies. It has been shown that at least 5–10% of the worker

force must be of the Bb genotype in order for Bb queens to be

accepted as new reproductives.(21) We speculate that the

recognition capability of workers that bear the b allele is

altered, allowing themajority ofBbqueens topassundetected.

In addition, the lack of intercolony discrimination in poly-

gyne colonies further reduces aggression towards non-

nestmate queens. The last factor is connected to queen

pheromone production and may explain why only Bb queens

are accepted into polygyne colonies. Fletcher and Blum(19)

showed that the weight of a queen is positively correlated with

the quantity of pheromone that she produces. It is therefore

possible that the reduced pheromone signal of the lighter Bb

queens (Fig. 2) is below the threshold that otherwise triggers

execution of BB queens.

Non-aggression towards Bb queens based on a weaker

queen pheromone signal seems likely an important factor,

but what is the exact role of the Bb workers that must be

present in colonies that acceptmultipleBb queens?How is the

presence of the presumed smelling-impaired Bb workers

preventing the rise of the otherwise aggressive collective?

Most importantly, however, how does this phenomenon relate

to the different allelic forms of GP-9? To address this issue,

we aim to determine the three-dimensional structure of the


BioEssays 27.1 97

three allelic protein variants in S. invicta. By determining the

structure of the proteins, we will learn whether the differences

in amino acid sequence translate into differences in protein

structure, identify the residues involved in binding the

pheromone ligand, and determine the shape of the binding

pocket. This may lead to predictions about the nature of the

unidentified ligand, as well as how these amino acid substitu-

tions affect its binding affinity. This will be crucial for inferring

the biochemical and behavioral mechanisms regulating

colony queen number, and for illuminating how variation in

Gp-9 genotype affects the process.

Finally, to further investigate the molecular mechanism of

social organization in the genus Solenopsis, we intend to

sequence additional polygyne populations of S. geminata

occurring elsewhere in its vast range todeterminewhetherGp-

9 sequence variation correspondswith polygyny in themanner

that we initially hypothesized or if reduced genetic diversity in

genes encoding recognition cues are consistently associated

in the history of these polygyne populations. In a second

approach, our aim is to examine Gp-9 genes from additional

species throughout the genus Solenopsis, with the purpose of

tracking the molecular evolutionary history of this fascinating

molecule.

Acknowledgments

I thank Lara Carroll, Ken Ross, AdamWilkins and two anony-

mous referees for helpful comments on the manuscript.

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to b or not to b: a pheromone-binding protein regulates colony social organization in fire ants

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