Has gene expression neofunctionalization in the fire ant antennae contributed to queen discrimination behavior?

Queen discrimination behavior in the fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. In contrast, monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles have suppressed recombination, determining which genes within the supergene mediate this differential worker behavior is difficult. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. To identify such genes, we sequenced RNA from four replicates of pooled antennae from three classes of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein‐coding genes with 13 encoding potential chemical metabolism or perception proteins. We focused on the two odorant perception genes: an odorant receptor SiOR463 and an odorant‐binding protein SiOBP12. We found that SiOR463 has been lost in the Sb genome. In contrast, SiOBP12 has an Sb‐specific duplication, SiOBP12b′, which is expressed in the SB/Sb worker antennae, while both paralogs are expressed in the body. Comparisons with another fire ant species revealed that SiOBP12b′ antennal expression is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.     

Simple inheritance,complex regulation: Supergene‐mediated fire ant queen polymorphism

The fire ant Solenopsis invicta exists in two alternate social forms: monogyne nests contain a single reproductive queen and polygyne nests contain multiple reproductive queens. This colony‐level social polymorphism corresponds with individual differences in queen physiology, queen dispersal patterns and worker discrimination behaviours, all evidently regulated by an inversion‐based supergene that spans more than 13 Mb of a “social chromosome,” contains over 400 protein‐coding genes and rarely undergoes recombination. The specific mechanisms by which this supergene influences expression of the many distinctive features that characterize the alternate forms remain almost wholly unknown. To advance our understanding of these mechanisms, we explore the effects of social chromosome genotype and natal colony social form on gene expression in queens sampled as they embarked on nuptial flights, using RNA‐sequencing of brains and ovaries. We observe a large effect of natal social form, that is, of the social/developmental environment, on gene expression profiles, with similarly substantial effects of genotype, including: (a) supergene‐associated gene upregulation, (b) allele‐specific expression and (c) pronounced extra‐supergene trans‐regulatory effects. These findings, along with observed spatial variation in differential and allele‐specific expression within the supergene region, highlight the complex gene regulatory landscape that emerged following divergence of the inversion‐mediated Sb haplotype from its homologue, which presumably largely retained the ancestral gene order. The distinctive supergene‐associated gene expression trajectories we document at the onset of a queen’s reproductive life expand the known record of relevant molecular correlates of a complex social polymorphism and point to putative genetic factors underpinning the alternate social syndromes.     

Sex investment ratios in monogyne and polygyne populations of the fire ant Solenopsis invicta

Both monogyne (single queen per colony) and polygyne (multiple queens per colony) populations of the fire ant Solenopsis invicta are good subjects for tests of kin selection theory because their genetic and reproductive attributes are well-characterized, permitting quantitative predictions about the degree to which sex investment ratios should be female-biased if workers and not queens control reproductive allocation. In the study populations, an investment ratio of 3 females: 1 male is predicted (a proportional investment in females of 0.75) in the monogyne form, whereas a proportional investment in females between 0.637 and 0.740 is expected in the polygyne form. To test these predictions, colonies from a single population of each social form were collected and censused during three different seasons. Consistent with their alternative modes of colony founding, monogyne colonies invested more in reproduction (sexual production) and less in growth/maintenance (worker production) than did the polygyne colonies. Overall, the sex investment ratios were female-biased in both forms, although there was considerable seasonal variation. After adjusting for sex-specific energetic costs, the proportional investment in females was 0.607 in the monogyne population, a value in between those expected under complete control by either the queen or the workers. However, when combined with data from four other previously studied monogyne populations in the U.S.A., the mean investment ratio did not differ significantly from the value predicted if workers have exclusive control. In the polygyne population, the proportional investment in females of 0.616 was consistent with the level of female bias expected under partial to complete worker control, although the potential influence of two confounding factors — possible contact with monogyne colonies and the preponderance of sterile diploid males — weakens this conclusion somewhat. Taken as a whole, the sex investment ratios of monogyne and polygyne populations of S. invicta are consistent with at least partial worker control. Of several ultimate and proximate explanations that have been proposed to explain inter-colonial variation in the sex investment ratio, only the effect of the primary sex ratio (female-determined eggs: male-determined eggs) laid by the queen appears to account for the observed variation among monogyne colonies. In the polygyne population, there is limited support for the hypothesis that greater resource abundance favors investment in females.     

Experimental Conversion of Colony Social Organization in Fire Ants (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>): Worker Genotype Manipulation in the Absence of Queen Effects

Colony social organization in the fire ant Solenopsis invicta appears to be under strong genetic control. In the invasive USA range, polygyny (multiple queens per colony) is marked by the presence of the Gp-9 ^b allele in most of a colony’s workers, whereas monogyny (single queen per colony) is associated with the exclusive occurrence of the Gp-9 ^B allele. Ross and Keller, Behav Ecol Sociobiol 51:287–295 (2002) experimentally manipulated social organization by cross-fostering queens into colonies of the alternate form, thereby changing adult worker Gp-9 genotype frequencies over time. Although these authors showed that social behavior switched predictably when the frequency of b-bearing adult workers crossed a threshold of 5–10%, the possibility that queen effects caused the conversions could not be excluded entirely. We addressed this problem by fostering polygyne brood into queenright monogyne colonies. All such treatment colonies switched social organization to become polygyne, coincident with their proportions of b-bearing workers exceeding 12%. Our results support the conclusion that polygyny in S. invicta is induced by a minimum frequency of colony workers carrying the b allele, and further confirm that its expression is independent of queen genotype or history, worker genotypes at genes not linked to Gp-9, and colony genetic diversity.     

The fire ant social supergene is characterized by extensive gene and transposable element copy number variation

In the fire ant Solenopsis invicta, a supergene composed of ~600 genes and having two variants, SB and Sb, regulates colony social form. In single queen colonies, all individuals carry only the SB allele, while in multiple queen colonies, some individuals carry the Sb allele. In this study, we characterized genes with copy number variation between SB and Sb‐carrying individuals. We showed extensive acquisition of gene duplicates in the Sb genome, with some likely involved in polygyne‐related phenotypes. We found 260 genes with copy number differences between SB and Sb, of which 239 have greater copy number in Sb. We observed transposable element (TE) accumulation on Sb, likely due to the accumulation of repetitive elements on the nonrecombining chromosome. We found a weak correlation between TE copy number and differential expression, suggesting some TEs may still be proliferating in Sb while many of the duplicated TEs have presumably been silenced. Among the 115 non‐TE genes with higher copy in Sb, enzymes responsible for cuticular hydrocarbon synthesis were highly represented. These include a desaturase and an elongase, both potentially responsible for differential queen odour and likely beneficial for polygyne ants. These genes seem to have translocated into the supergene from other chromosomes and proliferated by multiple duplication events. While the presence of TEs in supergenes is well documented, little is known about duplication of non‐TE genes and their possible adaptive role. Overall, our results suggest that gene duplications may be an important factor leading to monogyne and polygyne ant societies.     

Unexpected,well-developed nestmate recognition in laboratory colonies of polygyne imported fire ants (Hymenoptera: Formicidae)

Aggression bioassays were used to investigate nestmate recognition in polygyne laboratory colonies of the imported fire ant, Solenopsis invictaBuren. Unlike workers from polygyne field colonies, laboratory-maintained (>10 weeks) workers exhibited well-developed nestmate recognition. As in monogyne colonies of this species, both heritable and environmentally acquired (diet) odors provided recognition cues and were roughly additive in their effect. Within diet treatments, polygyne colonies responded in a graded fashion to polygyne conspecifics, monogyne conspecifics, and heterospecifics (S. richteri Forel),thus suggesting incipient genetic divergence between the two S. invictasocial forms. Hypotheses to account for the acute intraspecific discrimination observed in the laboratory are presented. Empirical testing of these hypotheses will illuminate ecological constraints and proximate mechanisms underlying the reduced intercolony discrimination associated with natural polygyne colonies of this and other ant species.     

The determinants of queen size in a socially polymorphic ant

In social animals, body size can be shaped by multiple factors, such as direct genetic effects, maternal effects, or the social environment. In ants, the body size of queens correlates with the social structure of the colony: colonies headed by a single queen (monogyne) generally produce larger queens that are able to found colonies independently, whereas colonies headed by multiple queens (polygyne) tend to produce smaller queens that stay in their natal colony or disperse with workers. We performed a cross‐fostering experiment to investigate the proximate causes of queen size variation in the socially polymorphic ant Formica selysi. As expected if genetic or maternal effects influence queen size, eggs originating from monogyne colonies developed into larger queens than eggs collected from polygyne colonies, be they raised by monogyne or polygyne workers. In contrast, eggs sampled in monogyne colonies were smaller than eggs sampled in polygyne colonies. Hence, eggs from monogyne colonies are smaller but develop into larger queens than eggs from polygyne colonies, independently of the social structure of the workers caring for the brood. These results demonstrate that a genetic polymorphism or maternal effect transmitted to the eggs influences queen size, which probably affects the social structure of new colonies.     

A simple genetic basis for complex social behaviour mediates widespread gene expression differences

A remarkable social polymorphism is controlled by a single Mendelian factor in the fire ant Solenopsis invicta. A genomic element marked by the gene Gp‐9 determines whether workers tolerate one or many fertile queens in their colony. Gp‐9 was recently shown to be part of a supergene with two nonrecombining variants, SB and Sb. SB/SB and SB/Sb queens differ in how they initiate new colonies, and in many physiological traits, for example odour and maturation rate. To understand how a single genetic element can affect all these traits, we used a microarray to compare gene expression patterns between SB/SB and SB/Sb queens of three different age classes: 1‐day‐old unmated queens, 11‐day‐old unmated queens and mated, fully reproductive queens collected from mature field colonies. The number of genes that were differentially expressed between SB/SB and SB/Sb queens of the same age class was smallest in 1‐day‐old queens, maximal in 11‐day‐old queens and intermediate in reproductive queens. Gene ontology analysis showed that SB/SB queens upregulate reproductive genes faster than SB/Sb queens. For all age classes, genes inside the supergene were overrepresented among the differentially expressed genes. Consistent with the hypothesized greater number of transposons in the Sb supergene, 13 transposon genes were upregulated in SB/Sb queens. Viral genes were also upregulated in SB/Sb mature queens, consistent with the known greater parasite load in colonies headed by SB/Sb queens compared with colonies headed by SB/SB queens. Eighteen differentially expressed genes between reproductive queens were involved in chemical signalling. Our results suggest that many genes in the supergene are involved in regulating social organization and queen phenotypes in fire ants.     

SIMPLE GENETIC BASIS FOR IMPORTANT SOCIAL TRAITS IN THE FIRE ANT SOLENOPSIS INVICTA

Variation in queen phenotype and reproductive role in the fire ant Solenopsis invicta has been shown to have a simple genetic basis in a single introduced population in the United States. The evidence consists of an association between this variation and queen genotype at Pgm-3, a phosphoglucomutase-encoding gene. In the present study, we surveyed Pgm-3 allele and genotype frequencies in diverse populations from the native and introduced ranges of this ant to learn whether this simple genetic basis for reproductive traits is a general feature of the species or a genetic anomaly in introduced ants stemming from a recent bottleneck or the invasion of novel habitats. No egg-laying queens living in polygyne (multiple-queen) nests possessed the homozygous genotype Pgm-3^a/a in any of the study populations, yet nonreproductive females from such nests (workers as well as queens that had not yet initiated oogenesis) possessed this genotype at moderate frequencies. Remarkably, Pgm-3^a/a was the most common genotype among all classes of females, including egg-laying queens, in monogyne (single-queen) nests from all populations studied. Genotype proportions at Pgm-3 in polygyne populations typically departed strongly from the proportions expected under Hardy-Weinberg equilibrium, whereas those in monogyne populations did not. These patterns establish that a single mendelian gene influences queen reproductive role in S. invicta and that this gene uniformly is under strong directional selection in the polygyne social form only. Moreover, the perfect association of Pgm-3 genotype and reproductive role in all populations, combined with the known function of phosphoglucomutase in insect metabolism, suggest that this gene may directly influence queen phenotypes rather than merely serving as a marker for a linked gene that causes the effects.     

The success of alternative reproductive tactics in monogyne populations of the ant Solenopsis invicta: significance for transitions in social organization

Newly produced queens from monogyne (single-queen) coloniesof the ant Solenopsis invicta usually initiate reproductionindependently, that is, without worker assistance. However,some recently mated queens attempt to bypass this risky phaseof new colony foundation by entering established nests to reproduce,although it is unclear how often these queens are successfulin natural populations. We surveyed a mature monogyne populationof S. invicta in both 1995 and 1996 for colonies headed by queensincapable of independent colony founding (diploid-male-producingqueens) in order to estimate the frequency of colonies thatare headed by queens that initiated reproduction within establishednests (adopted queens). Using the frequency of diploid-male-producingqueens among the recently mated queens in this population, weestimated that the overall rate of queen replacement by adoptedqueens is about 0.7% per colony per year. Although theory suggeststhat a change to a novel queen reproductive tactic could beassociated with a fundamental change in social organization(queen number), this does not appear to be the case in monogyneS. invicta. However, the evolution of nest-infiltrating reproductivetactics by queens in a monogyne population and the evolutionof multiple-queen societies may result from similar ecologicalpressures facing newly mated queens. We therefore incorporatethis strategy into an existing theoretical framework that wasdeveloped to explain the evolution of alternative social organizationsin ants, providing testable predictions regarding the distributionand frequency of queen adoption in other single-queen ant societies.     

Sources of nestmate recognition cues in the imported fire ant Solenopsis invicta buren (Hymenoptera: Formicidae)

Aggression bioassays were conducted to investigate nestmate (worker-worker) recognition in monogyne colonies of the imported fire ant. Environmentally correlated cues dominated the recognition cue hierarchy of laboratory and field colonies, anddiet alone significantly modified recognition labels and templates of laboratory-reared workers. ‘Discriminators’ associated with worker genotype also affected recognition, but ‘queen discriminators’ did not significantly affect either labels or templates of laboratoryreared workers exposed as adults to alien queens for 28 days. Factors contributing to the importance of environmentally derived recognition cues in this species and the potential implications of such cues for the formation of polygynous fire ant populations are discussed.     

Multiple virus infections occur in individual polygyne and monogyne Solenopsis invicta ants

Concurrent infections of Solenopsis invicta colonies with S. invicta virus 1 (SINV-1), SINV-2, and SINV-3 has been reported. However, whether individual ants were capable of supporting multiple virus infections simultaneously was not known, nor whether the social form of the colony (polygyne or monogyne) had an influence on the occurrence of multiple infection rates in individual ants. S. invicta field populations were sampled sequentially to establish whether multiple virus infections co-occurred in individual worker ants. In addition, the intra-colony virus infection rates were compared in monogyne and polygyne field colonies to determine whether social form played a role in the viral infection prevalence. All combinations of virus infection (SINV-1, SINV-2, or SINV-3 alone, SINV-1 & SINV-2, SINV-1 & SINV-3, SINV-2 & SINV-3, and SINV-1, SINV-2 & SINV-3) were detected in individual worker ants as well as queens in the field. Thus, individual S. invicta ants can be infected simultaneously with all combinations of the S. invicta viruses. Colony social form did have an influence on the intra-colony prevalence of multiple S. invicta virus infections. Polygyne colonies exhibited significantly greater intra- and inter-colony single and multiple virus infections compared with monogyne colonies.     

Relationship of queen number and worker size in polygyne colonies of the fire antSolenopsis invicta

Summary We examined the relationship between queen number and worker size in colonies of the fire antSolenopsis invicta. Worker size in monogyne colonies was significantly greater than in polygyne colonies; furthermore, polygyne colonies snowed a strong negative linear relationship between queen number and worker size. Higher queen pheromone level and/or decreased food availability accompanying an increase in queen number likely play important roles in producing the observed patterns.     

A comparison of queen oviposition rates from monogyne and polygyne fire ant, Solenopsis invicta, colonies

Abstract. The oviposition rate of individual queens of Solenopsis invicta Buren (Hymenoptera: Formicidae) in relation to their weight and number of queens present in the colony was investigated by direct 2 h observations. There is a strong positive correlation between the weight of a queen and its oviposition rate in both monogyne and polygyne colonies. However, the number of eggs laid per mg queen is higher for moonogyne queens than for polygyne queens. This difference is more evident when the total weight of queens present in a colony is considered. The individual queen oviposition rate is negatively correlated with the number of queens in the colony. In addition, the weight loss per egg laid is significantly greater for polygyne than for monogyne queens, probably due to differences in egg size. These data suggest that oviposition is more efficient in monogyne than in polygyne queens at the individual level; however, at the colony level, polygyne colonies produce significantly more eggs. Comparison of colony level efficiency predicts that polygyne colonies must have at least nine queens to compete reproductively with a mature monogyne queen. Therefore, oligogyny does not appear to be a viable strategy for S.invicata.     

Genetic determination of female castes in a hybridogenetic desert ant

In most social insects, the brood is totipotent and environmental factors determine whether a female egg will develop into a reproductive queen or a functionally sterile worker. However, genetic factors have been shown to affect the female's caste fate in a few ant species. The desert ant Cataglyphis hispanica reproduces by social hybridogenesis. All populations are characterized by the coexistence of two distinct genetic lineages. Queens are almost always found mated with a male of the alternate lineage than their own. Workers develop from hybrid crosses between the genetic lineages, whereas daughter queens are produced asexually via parthenogenesis. Here, we show that the association between genotype and caste in this species is maintained by a ‘hard‐wired’ genetic caste determination system, whereby nonhybrid genomes have lost the ability to develop as workers. Genetic analyses reveal that, in a rare population with multiple‐queen colonies, a significant proportion of nestmate queens are mated with males of their own lineage. These queens fail to produce worker offspring; they produce only purebred daughter queens by sexual reproduction. We discuss how the production of reproductive queens through sexual, intralineage crosses may favour the stability of social hybridogenesis in this species.     

Alternative genetic foundations for a key social polymorphism in fire ants

Little is known about the genetic foundations of colony social organization. One rare example in which a single major gene is implicated in the expression of alternative social organizations involves the presumed odorant-binding protein gene Gp-9 in fire ants. Specific amino acid substitutions in this gene invariably are associated with the expression of monogyny (single queen per colony) or polygyny (multiple queens per colony) in fire ant species of the Solenopsis richteri clade. These substitutions are hypothesized to alter the abilities of workers to recognize queens and thereby regulate their numbers in a colony. We examined whether these same substitutions underlie the monogyny/polygyny social polymorphism in the distantly related fire ant S. geminata. We found that Gp-9 coding region sequences are identical in the polygyne and monogyne forms of this species, disproving our hypothesis that one or a few specific amino acid replacements in the protein are necessary to induce transitions in social organization in fire ants. On the other hand, polygyne S. geminata differs genetically from the monogyne form in ways not mirrored in the two forms of S. invicta, a well-studied member of the S. richteri clade, supporting the conclusion that polygyny did not evolve via analogous routes in the two lineages. Specifically, polygyne S. geminata has lower genetic diversity and different gene frequencies than the monogyne form, suggesting that the polygyne form originated via a founder event from a local monogyne population. These comparative data suggest an alternative route to polygyny in S. geminata in which loss of allelic variation at genes encoding recognition cues has led to a breakdown in discrimination abilities and the consequent acceptance of multiple queens in colonies.     

Venom alkaloids of monogyne and polygyne forms of the red imported fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>, in Taiwan

Both monogyne and polygyne colonies of Solenopsis invicta now occupy Taiwan. Although venom alkaloids of these ants have been described and synthesized, we here report on a quantitative analysis of the two social forms for the first time. The alkaloids were studied by gas chromatography coupled to mass spectrometry (GC-MS), and six major venom alkaloids were detectable in both types of workers. Both C₁₃:C_13:1 and C₁₅:C_15:1 ratios in alkaloid venom of monogyne workers were statistically higher than that of polygyne workers, but the sum of proportions of unsaturated alkaloids of polygyne workers was significantly higher than that of monogyne workers, regardless of growth temperature, sampling seasons or geographic location. Results of this study demonstrate that the difference in the proportions of unsaturated alkaloids and the ratios of C₁₃:C_13:1 and C₁₅:C_15:1 alkaloids might be a good indicator for differentiating monogyne and polygyne forms of S. invicta. Received 20 February 2008; revised 4 July 2008; accepted 5 August 2008.     

A TEST OF QUEEN RECRUITMENT MODELS USING NUCLEAR AND MITOCHONDRIAL MARKERS IN THE FIRE ANT SOLENOPSIS INVICTA

We assess nestmate queen relatedness and the genetic similarity of neighboring nests in the polygyne (multiple-queen) social form of the introduced fire ant Solenopsis invicta using both nuclear and mitochondrial markers. We find that estimates of queen relatedness calculated with both types of markers do not differ statistically from zero. Furthermore, there is no significant relationship between the genetic similarity and geographic proximity of nests in each of six study sites. In contrast to these findings, sites show strong mitochondrial, but no nuclear, genetic differentiation. Our results suggest that nonnestmate queen recruitment occurs at a high frequency in introduced populations of this species. Moreover, queens within nests seem to represent a random sample of the queens within the site in which they reside. Therefore, kin selection models that rely on the recruitment of only nestmate queens to explain the persistence of polygyny in ants do not apply to polygyne S. invicta in its introduced range.     

Effect of mono- and polygyne social forms on transmission and spread of a microsporidium in fire ant populations

Thelohania solenopsae is a pathogen of the red imported fire ant, Solenopsis invicta, which debilitates queens and eventually causes the demise of colonies. Reductions of infected field populations signify its potential usefulness as a biological control agent. Thelohania solenopsae can be transmitted by introducing infected brood into a colony. The social forms of the fire ant, that is, monogyny (single queen per colony) or polygyny (multiple queens per colony), are associated with different behaviors, such as territoriality, that affect the degree of intercolony brood transfer. T. solenopsae was found exclusively in polygyne colonies in Florida. Non-synchronous infections of queens and transovarial transmission favor the persistence and probability of detecting infections in polygynous colonies. However, queens or alates with the monogyne genotype can be infected, and infections in monogyne field colonies have been reported from Louisiana and Argentina. Limited independent colony-founding capability and shorter dispersal of alate queens with the polygyne genotype relative to monogyne alates may facilitate the maintenance of infections in local polygynous populations. Demise of infected monogyne colonies can be twice as fast as in polygyne colonies and favors the pathogen's persistence in polygyne fire ant populations. The social form of the fire ant reflects different physiological and behavioral aspects of the queen and colony that will impact T. solenopsae spread and ultimate usefulness for biological control.     

Unusual Behavior of Polygyne Fire Ant Queens on Nuptial Flights

This study reports previously undescribed behavior of fire ant queens (Solenopsis invicta) on their nuptial flights. We captured large numbers of alate (winged) queens flying at low altitudes in dense swarms that were virtually devoid of males. We assayed the genotypes of these alate queens at the locus Gp-9, which exhibits strong genotype frequency differences between monogyne (single-queen) and polygyne (multiple-queen) populations, and found that almost all of these low-flying queens originated from polygyne colonies. Comparisons of mtDNA haplotype distributions of these queens to those of alates leaving polygyne nests suggest that the flying queens had not dispersed more than a few hundred meters. Moreover, the proportion of flying queens that were mated did not differ significantly from the proportion of reproductive queens that were mated within the same sites. Thus the flight behavior appears to occur subsequent to mating. We suggest that the flying queens are sampling the local environment in order to select a suitable landing site. Such a site would contain established polygyne nests into which the queens may be adopted as new reproductives.