The Molecular Clockwork of the Fire Ant Solenopsis invicta

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.     

GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant,Solenopsis invicta

The red imported fire ant (RIFA), Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.     

Regulation of Diet in the Fire Ant, Solenopsis invicta

In social insects, colony nutrition depends upon the volume and quality of food distributed, ingested, and assimilated by its members. The ability of Solenopsis invicta workers and larvae to regulate the volume of food ingested individually has been well documented. In this paper, the ability of fire ant workers and larvae to regulate the quality and type of food ingested is demonstrated. Larvae displayed independent appetites for solid protein, amino acid solution, and sucrose solution. When larvae that had fed on one food type were switched to another, they fed on the second food type at rates characteristic of that food type, not of the volume of food previously ingested. Larvae preferred concentrated sucrose and amino acids solutions over dilute solutions. Larval fullness was thus a relative property, depending upon the nature of food as well as the volume ingested. The number of workers recruiting to food sites also depended upon food concentration and food type. Workers preferred sucrose to amino acids, concentrated to dilute solutions, and novel to accustomed food. The absence of protein in the worker diet rather than the presence of larvae caused workers to switch their preference from sugar to amino acids solutions. When the colony was offered sucrose and amino acids solutions simultaneously, individual workers ingested from one or the other site, but not both. Little mixing of crop contents occurred when workers solicited from one another inside the nest. Workers tended to regurgitate to larvae after ingesting amino acids and to other workers after ingesting sucrose. The mechanism regulating the distribution of protein pellets, which workers do not ingest, among larvae is unknown. In summary, colony nutrition was regulated by a chain of demand. Forager hunger determined the rate at which food flowed from the environment into the nest. Larval hunger and nest-worker hunger determined the rate and direction in which food moved within the nest.     

Polyphasic Wake/Sleep Episodes in the Fire Ant, Solenopsis Invicta

Sleep is a well-studied biological process in vertebrates, particularly birds and mammals. Less is know about sleep in solitary and social invertebrates, particularly the ants. This paper reports a study of light/dark periods on worker activity as well as sleep location, posture and the wake/sleep cycles of fire ant workers and queens located in an artificial nest chamber. Workers slept in one of three locations: on the ceiling, against the chamber wall or in the center of the chamber floor. Workers on the ceiling or against the chamber wall slept for longer periods than those at the center of the chamber floor where most grooming and feeding activity occurred. When sleeping, queens huddled together. Their close contact generated synchronized wake/sleep cycles with each other. Sleep posture was distinctly different than wake posture. During deep sleep, queens and workers folded their antennae and were non-responsive to contact by other ants. Another indicator of deep sleep was rapid antennal movement (RAM sleep). Sleep episodes were polyphasic. Queens averaged ~92 sleep episodes per day, each episode lasting ~6 min, for a total of ~9.4 h of sleep per day. Workers averaged ~253 sleep episodes lasting 1.1 min each for a total of ~4.8 h of sleep per day. Activity episodes were unaffected by light/dark periods. Workers were hypervigilant with an average of 80% of the labor force completing grooming, feeding or excavation tasks at any given time. These findings reinforce the parental exploitation hypothesis—sterile workers are a caste of disposable, short-lived helpers whose vigilance and hyperactivty increases the queen’s fitness by buffering her and her fertile offspring from environmental stresses.     

Proteomic View of the Venom from the Fire Ant Solenopsis invicta Buren

Fire ants are well-known by their aggressive stinging behavior, causing many stinging incidents of medical importance. The limited availability of fire ant venom for scientific and clinical uses has restricted, up to now, the knowledge about the biochemistry, immunology, and pharmacology of these venoms. For this study, S. invicta venom was obtained commercially and used for proteomic characterization. For this purpose, the combination of gel-based and gel-free proteomic strategies was used to assign the proteomic profile of the venom from the fire ant S. invicta. This experimental approach permitted the identification of 46 proteins, which were organized into four different groups according to their potential role in fire ant venom: true venom components, housekeeping proteins, body muscle proteins, and proteins involved in chemical communication. The active venom components that may not present toxic roles were classified into three subgroups according to their potential functions: self-venom protection, colony asepsis, and chemical communication. Meanwhile, the proteins classified as true toxins, based on their functions after being injected into the victims' bodies by the fire ants, were classified in five other subgroups: proteins influencing the homeostasis of the victims, neurotoxins, proteins that promote venom diffusion, proteins that cause tissue damage/inflammation, and allergens.     

Potential Global Range Expansion of the Invasive Fire Ant, Solenopsis invicta

The red imported fire ant, Solenopsis invicta Buren, is an invasive pest that has become widespread in the southern United States and Caribbean after introduction from South America in the 1930s. This species, which has diverse detrimental impacts on recipient communities, was recently discovered in Australia and New Zealand and has the potential to colonize numerous other regions. We used a dynamic, ecophysiological model of colony growth to predict the potential global range expansion of this invasive species. Based on minimum and maximum daily temperatures, the model estimates colony alate production and predicts future geographic range limits. Because S. invicta populations are limited by arid conditions as well as cold temperatures, we superimposed precipitation data upon temperature-based predictions, to identify regions that do not receive enough rainfall to support this species across the landscape. Many areas around the globe, including large portions of Europe, Asia, Africa, Australia, and numerous island nations, are at risk for S. invicta infestation. Quarantine officials should be vigilant for any accidental introductions of this pest in susceptible regions. Costs of eradication increase dramatically as the area of infestation grows, and large infestations may be impossible to eradicate. Other South American Solenopsis fire ants (e.g., S. richteri Forel) may become invasive if the opportunity arises, and our predictions for S. invicta may approximate the potential range limits for these species as well.     

Ontogeny of the Defensive Stinging Behavior of the Fire Ant, Solenopsis invicta

The fire ant, Solenopsis invicta, appears to deviate from the usual age-related pattern of defensive behavior seen in other social insects; instead of older workers being more defensive than younger ones, they are less so. Here I test this pattern by quantifying changes in the defensive stinging behavior of S. invicta workers of known age. I found defensiveness, measured as both the number of stings delivered and the amount of venom delivered per sting (venom dose), to increase with age initially but then decline after a mid-age peak. This hump-shaped ontogeny may be the result of S. invicta's foraging strategy, which effectively shifts the responsibility of nest defense to workers younger than foraging age. It is S. invicta's mid-aged workers that are the most defensive, probably because they are both physiologically and spatially the most suitable nest defenders.     

Tetradonema solenopsis n. sp. (Nematoda: Tetradonematidae) Parasitic on the Red Imported Fire Ant Solenopsis invicta Buren from Brazil

Explorations in Brazil to find parasites of the red imported fire ant, Solenopsis invicta Buren, have uncovered a new species of the Tetradonematidae, Tetradonema solenopsis n. sp. The nematode parasite was fatal to about 25% of the colony. The female nematodes are large and sausage shaped and the males are small and difficult to find, which is typical of the genus. The ant is a new host record for tetradonematids.     

Mating Behavior and Colony Founding of the Slave-Making Ant Formica sanguinea (Hymenoptera: Formicidae)

The mating and postmating behavior of reproductives belonging to two sympatric dulotic colonies of the facultative slave-making ant Formica sanguinea was analyzed in the field. Our observations showed that the European blood-red ant adopts a reproductive behavior similar to the male aggregation syndrome. Newly mated females return to a dulotic colony and often wait for a raid. Following a slave raid is an advantageous strategy to locate and invade host nests and to establish a new dulotic colony. In the laboratory, the following modes of colony founding were studied: independent, adoption, alliance, usurpation, and brood raiding. Independent foundation was possible only when several females were kept together. Alliance was obtained with females of two potential slave species (F. cunicularia, F. rufibarbis). Usurpation and adoption were more frequent in the incipient than in the mature host colonies. Mixed colonies were always obtained after the sack of the host pupae. It seems likely that, rather than conspecific adoption followed by budding, F. sanguinea relies on temporary parasitism to start new colonies.     

Colony Wide Behavioral Contexts of Stridulation in Imported Fire Ants (Solenopsis invicta Buren)

Red imported fire ants, Solenopsis invicta, possess stridulatory organs and stridulate in a variety of contexts. We used a stethoscope mounted microphone to study stridulation at the colony level in the context of emigration, disturbance, and excavation. In conjunction with preliminary observations of nest and foraging activities, our results suggest stridulation serves multiple functions in S. invicta. Stridulation was not significantly increased in colonies during responses to disturbance, and only marginally during colony emigration. Colonies involved in excavation, however, exhibited a significant increase in stridulatory activity. Four possible explanations for the function of stridulation in this context are discussed in relation to the stridulatory behavior of individuals, solitary wasps, and published literature on formicid stridulation.     

Effect of a Founder Event on Variation in the Genetic Sex-Determining System of the Fire Ant Solenopsis Invicta

Effects of a recent founder event on genetic diversity in wild populations of the fire ant Solenopsis invicta were studied, with particular attention given to the genetic sex-determining system. Diploid males are far more common relative to haploid males in introduced populations than in native populations of fire ants, and queens that produce diploid males account for a significantly larger proportion of the mated queens in introduced than in native populations. Differences between native and introduced populations in attributes of the mating systems (i.e., queen mating frequency or level of inbreeding) can be excluded as factors contributing to these different levels of diploid male production. Thus, we conclude that diploid males have increased in frequency in introduced populations because of a loss of allelic diversity at the sex-determining locus (loci). This loss of sex alleles has generated a substantial increase in the estimated segregational genetic load associated with production of sterile diploid males in introduced populations over the load in native populations. The loss of allelic diversity in the sex-determining system in introduced S. invicta is paralleled by a loss of electrophoretically detectable rare alleles at protein-encoding loci. Such concordance between these different types of markers is predicted because each of the many sex alleles present in the native populations is expected to be rare. Estimates of expected heterozygosity (H(exp)) based on 76 electrophoretic loci do not differ significantly between the native and introduced fire ant populations, illustrating the lack of sensitivity of this measure for detecting many types of bottlenecks.     

Visual Navigation during Colony Emigration by the Ant Temnothorax rugatulus

Many ants rely on both visual cues and self-generated chemical signals for navigation, but their relative importance varies across species and context. We evaluated the roles of both modalities during colony emigration by Temnothorax rugatulus. Colonies were induced to move from an old nest in the center of an arena to a new nest at the arena edge. In the midst of the emigration the arena floor was rotated 60°around the old nest entrance, thus displacing any substrate-bound odor cues while leaving visual cues unchanged. This manipulation had no effect on orientation, suggesting little influence of substrate cues on navigation. When this rotation was accompanied by the blocking of most visual cues, the ants became highly disoriented, suggesting that they did not fall back on substrate cues even when deprived of visual information. Finally, when the substrate was left in place but the visual surround was rotated, the ants'' subsequent headings were strongly rotated in the same direction, showing a clear role for visual navigation. Combined with earlier studies, these results suggest that chemical signals deposited by Temnothorax ants serve more for marking of familiar territory than for orientation. The ants instead navigate visually, showing the importance of this modality even for species with small eyes and coarse visual acuity.     

Genetic Transformation of Midgut Bacteria from the Red Imported Fire Ant (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>)

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37°C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants. Freder Medina and Haiwen Li have contributed equally to this work.     

Distribution of Endosymbiotic Reproductive Manipulators Reflects Invasion Process and Not Reproductive System Polymorphism in the Little Fire Ant Wasmannia auropunctata

Endosymbiotic reproductive manipulators may have drastic effects on the ecological and evolutionary dynamics of their hosts. The prevalence of these endosymbionts reflects both their ability to manipulate their hosts and the history of the host populations. The little fire ant Wasmannia auropunctata displays a polymorphism in both its reproductive system (sexual versus clonal populations) and the invasive status of its populations (associated to a habitat shift). We first screened for the presence of a diverse array of reproductive parasites in sexual and clonal populations of W. auropunctata, as a means to investigate the role of endosymbionts in reproductive phenotypes. Wolbachia was the only symbiont found and we then focused on its worldwide distribution and diversity in natural populations of W. auropunctata. Using a multilocus scheme, we further characterized the Wolbachia strains present in these populations. We found that almost all the native sexual populations and only a few clonal populations are infected by Wolbachia. The presence of similar Wolbachia strains in both sexual and clonal populations indicates that they are probably not the cause of the reproductive system polymorphism. The observed pattern seems rather associated to the invasion process of W. auropunctata. In particular, the observed loss of Wolbachia in clonal populations, that recurrently emerged from sexual populations, likely resulted from natural heat treatment and/or relaxed selection during the shift in habitat associated to the invasion process.     

Insecticidal Activity of Piper Essential Oils from the Amazon Against the Fire Ant Solenopsis saevissima (Smith) (Hymenoptera: Formicidae)

Pepper plants in the genus Piper (Piperales: Piperaceae) are common in the Brazilian Amazon and many produce compounds with biological activity against insect pests. We evaluated the insecticidal effect of essential oils from Piper aduncum, Piper marginatum (chemotypes A and B), Piper divaricatum and Piper callosum against workers of the fire ant Solenopsis saevissima (Smith) (Hymenoptera: Formicidae), as well as their chemical composition by gas chromatography and gas chromatography?Cmass spectrometry. The lowest median lethal concentration (LC₅₀) in 48?h was obtained with the oil of P. aduncum (58.4?mg/L), followed by the oils of P. marginatum types A (122.4?mg/L) and B (167.0?mg/L), P. divaricatum (301.7?mg/L), and P. callosum (312.6?mg/L). The major chemical constituents were dillapiole (64.4%) in the oil of P. aduncum; p-mentha-1(7),8-diene (39.0%), 3,4-methylenedioxypropiophenone (19.0%), and (E)-??-ocimene (9.8%) in P. marginatum chemotype A and (E)-isoosmorhizole (32.2%), (E)-anethole (26.4%), isoosmorhizole (11.2%), and (Z)-anethole (6.0%) in P. marginatum chemotype B; methyleugenol (69.2%) and eugenol (16.2%) in P. divaricatum; and safrole (69.2%), methyleugenol (8.6%), and ??-pinene (6.2%) in P. callosum. These chemical constituents have been previously known to possess insecticidal properties.     

Cloning and Expression of Multiple Cytochrome P450 Genes: Induction by Fipronil in Workers of the Red Imported Fire Ant (Solenopsis invicta Buren)

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the “first line of defense” when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL⁻¹ (median lethal dose) and 0.56 μg mL⁻¹ (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.