Sanitizing the fortress: protection of ant brood and nest material by worker antibiotics

Social groups are at particular risk for parasite infection, which is heightened in eusocial insects by the low genetic diversity of individuals within a colony. To combat this, adult ants have evolved a suite of defenses to protect each other, including the production of antimicrobial secretions. However, it is the brood in a colony that are most vulnerable to parasites because their individual defenses are limited, and the nest material in which ants live is also likely to be prone to colonization by potential parasites. Here, we investigate in two ant species whether adult workers use their antimicrobial secretions not only to protect each other but also to sanitize the vulnerable brood and nest material. We find that, in both leaf-cutting ants and weaver ants, the survival of the brood was reduced and the sporulation of parasitic fungi from them increased, when the workers nursing them lacked functional antimicrobial-producing glands. This was the case for both larvae that were experimentally treated with a fungal parasite (Metarhizium) and control larvae which developed infections of an opportunistic fungal parasite (Aspergillus). Similarly, fungi were more likely to grow on the nest material of both ant species if the glands of attending workers were blocked. The results show that the defense of brood and sanitization of nest material are important functions of the antimicrobial secretions of adult ants and that ubiquitous, opportunistic fungi may be a more important driver of the evolution of these defenses than rarer, specialist parasites.     

Colonial recognition of fungus in the fungus-growing ant Acromyrmex subterraneus subterraneus (Hymenoptera: Formicidae)

Summary. Leaf cutting ants live in symbiosis with a basidiomycete fungus that is exploited as a source of nutrients for the ant larvae. Tests of fungus transport demonstrated that Acromyrmex subterraneus subterraneus workers discriminate concolonial fungus from alien fungus, and rejected the latter. Larvae and pupae of the ant were used as controls. Chemical analysis of the fungus revealed a great similarity between its hydrocarbon profile and that found on the ant brood. Experiments with lures showed that chemical extracts from the fungus are responsible for this discrimination process. Moreover, the presence of brood inside the fungus seemed to be important for discrimination of the fungus by workers. Resident workers accepted concolonial broodless fungus less than concolonial fungus inoculated with brood odor. Fungus seems to acquire colonial odor passively, simply by contact with the brood. The impact of fungus volume present in the nest on closure of the colony is discussed. We show here for the first time the importance of a symbiotic vegetal organism in colonial recognition in social insects. Received 14 April 2000; accepted 29 September 2000     

Larval hemolymph feeding in the ant Leptanilla japonica by use of a specialized duct organ,the “larval hemolymph tap” (Hymenoptera: Formicidae)

Summary The larvae of the migratory Japanese ant Leptanilla japonica Baroni Urbani have a specialized duct organ on each side of the 4th abdominal segment. Behavioral and histological studies have shown that the adult ants are able to imbibe hemolymph directly from the larval body cavity through these organs, each of which is referred to here as a larval hemolymph tap. Laboratory observations further confirm that larval hemolymph feeding (LHF) is the sole source of nutrient for the queens. L. japonica is cyclical in its brood production. All larvae in a colony develop in concert, and when they are mature, the queen performs much active LHF on them. Nourished in this way, she achieves full physogastry within a period of only a few days and then lays a batch of 100–200 eggs. LHF thus facilitates synchronization of brood maturation and concentrated production of eggs by the queen. The larval hemolymph tap has probably evolved de novo in Leptanilla, in relation to cyclical brood production by species with small colony populations. Obviously, the function of this larval organ is non-selfish, contributing to overall colony-level functioning. The larvae of Leptanilla are thus properly qualified as a distinct class of caste in ant societies.     

Task selection by workers of the fire ant Solenopsis invicta

The effects of worker size, age, and crop fullness on the flow of food into the colony were assessed using video recording and playback. Regardless of the level of colony satiation, small workers seldom had full crops and were more involved in larval grooming than in food traffic. Large workers played little role in larval care, but tended to be recruited easily to a food source and to store food in their crops. Medium workers had crops ranging from empty to full because they alternated between ingesting from and donating food to other colony members. Medium workers were the most versatile, engaging competently in food recruitment, larval grooming, and larval feeding. They displayed considerable variation in the frequency at which they fed larvae: some fed a few larvae before switching to other tasks, others fed over a hundred larvae before switching. The persistence, or lack thereof, of a worker's feeding response suggests a flexibility unaccounted for by the fixed-threshold-response hypothesis. Worker coverage of the brood pile was a dynamic equilibrium process unaffected by worker size, age, or crop fullness, or by differences in the nutritional or hygienic states of larvae. In summary, it appeared that worker size and age offered coarse regulation of task selection by workers, whereas crop fullness, flexible response, and task switching fine-tuned task selection. Received: 25 May 1998 / Accepted after revision: 20 August 1998     

Age-related repertoire expansion and division of labor in Pheidole dentata (Hymenoptera: Formicidae): a new perspective on temporal polyethism and behavioral plasticity in ants

The controversy concerning the extent to which the organization of division of labor in social insects is a developmental process or is based on task allocation dynamics that emerge from colony need independent of worker age and endocrine or neural state has yet to be resolved. We present a novel analysis of temporal polyethism in the ant Pheidole dentata, demonstrating that task attendance by minor workers does not shift among spatially associated sets of behaviors that minimally overlap but rather expands with age. Our results show that the number of tasks performed by older minors increases through the addition and retention of behaviors, with up to a sixfold increase in repertoire size from day 1 to day 20 of adult life. We also show that older minors respond to colony needs by performing significantly more brood care as its demand increases, indicating that they can quickly upregulate nursing according to labor requirements. This level of plasticity was absent in younger siblings. The breadth of responsiveness to task-related olfactory stimuli increased with age. In a binary choice test in which young and old minor workers could orient toward odorants from brood or food, older workers responded to both brood and food, whereas young workers responded only to brood. These dissimilar responses to stimuli associated with nursing and foraging indicate age-related differences in sensory ability and provide a physiological basis for the age-related repertoire expansion model. We discuss repertoire expansion in P. dentata in light of behavioral development and caste flexibility in ants.     

High social density increases foraging and scouting rates and induces polydomy in Temnothorax ants

Many organisms live in crowded groups where social density affects behavior and fitness. Social insects inhabit nests that contain many individuals where physical interactions facilitate information flow and organize collective behaviors such as foraging, colony defense, and nest emigration. Changes in nest space and intranidal crowding can alter social interactions and affect worker behavior. Here, I examined the effects of social density on foraging, scouting, and polydomy behavior in ant colonies—using the species Temnothorax rugatulus. First, I analyzed field colonies and determined that nest area scaled isometrically with colony mass—this indicates that nest area changes proportionally with colony size and suggests that ants actively control intranidal density. Second, laboratory experiments showed that colonies maintained under crowded conditions had greater foraging and scouting activities compared to the same colonies maintained at a lower density. Moreover, crowded colonies were significantly more likely to become polydomous. Polydomous colonies divided evenly based on mass between two nests but distributed fewer, heavier workers and brood to the new nests. Polydomous colonies also showed different foraging and scouting rates compared to the same colonies under monodomous conditions. Combined, the results indicate that social density is an important colony phenotype that affects individual and collective behavior in ants. I discuss the function of social density in affecting communication and the organization of labor in social insects and hypothesize that the collective management of social density is a group level adaptation in social insects.     

Brood sorting by ants: distributing the workload over the work-surface

Summary Leptothorax unifasciatus ant colonies occupy flat crevices in rocks in which their brood is kept in a single cluster. In artificial nests made from two glass plates sandwiched together, designed to mimic the general proportions of their nest sites in the field, such colonies arrange their brood in a distinct pattern. These patterns may influence the priority with which different brood are tended, and may therefore influence both the division of labour and colony demography. Different brood stages are arranged in concentric rings in a single cluster centred around the eggs and micro-larvae. Successively larger larvae are arranged in progressive bands away from the centre of the brood cluster. However, the largest and oldest brood items, the prepupae and pupae, are placed in an intermediate position between the largest and most peripheral larvae and the larvae of medium size. Dirichlet tessellations are used to analyze these patterns and show that the tile areas, the area closer to each item than its neighbours, allocated to each type of item increase with distance from the centre of the brood cluster. There is a significant positive correlation between such tile areas and the estimated metabolic rates of each type of brood item. The ants may be creating a domain of care around each brood item proportional to that item's needs. If nurse workers tend to move to the brood item whose tile they happen to be within when they have care to donate, they may apportion such care according to the needs of each type of brood. When colonies emigrate to new nests they rapidly recreate these characteristic brood patterns.     

The social behavior of Leptothorax allardycei (Hymenoptera,Formicidae): time budgets and the evolution of worker reproduction

Summary The use of time by workers of the ant Leptothorax allardycei (Mann) is examined. Theoretical predictions are developed concerning the maximal lowering in colony reproductive output that is consistent with the evolution of worker production. Measurements are made of the effect of aggression on colony efficiency. Analysis of time budgets indicate that a typical ant spends a large fraction (0.55) of its time quiescent and another large fraction of time (0.32) involved in undifferentiated activity. Dominance activity and brood care together make up about 11% of the total time. The amount of time spent on dominance activity is negatively related to the amount of time spent on brood care, but positively related to the amount of time that an ant is active. The amount of time that an ant has available for brood care which is actually spent on brood care declines with the amount of time spent on dominance activity. The amount of time that a worker spends feeding liquid food to larvae is a function of hierarchy rank; alpha spends the most time, beta less and gamma still less. The spread of the trait of worker reproduction is examined theoretically, with particular regard to the associated costs to colony reproduction. Worker reproduction can spread through a population, under a variety of formulations, provided the cost to colony reproduction is less than some critical value in the neighborhood of 0.17–0.22 of the total colony output. The cost of worker reproduction in L. allardycei is estimated in two ways: as a time cost and as a reduction in the total number of brood tended per unit time. The two estimates of cost are 0.15 and 0.13 respectively. The reproductive options of the worker caste and the division of reproductive labor vary considerably between species. Reproduction by workers yields fitness differences between workers and results in competition among workers with the result that colony efficiency is affected.     

Social influences on body size and developmental time in the bumblebee Bombus terrestris

In many social insects, including bumblebees, the division of labor between workers relates to body size, but little is known about the factors influencing larval development and final size. We confirmed and extend the evidence that in the bumblebee Bombus terrestris the adult bee body size is positively correlated with colony age. We next performed cross-fostering experiments in which eggs were switched between incipient (before worker emergence) and later stage colonies with workers. The introduced eggs developed into adults similar in size to their unrelated nestmates and not to their same-age full sisters developing in their mother colony. Detailed observations revealed that brood tending by the queen decreases, but does not cease, in young colonies with workers. We next showed that both worker number and the queen presence influenced the final size of the developing brood, but only the queen influence was mediated by shortening developmental time. In colonies separated by a queen excluder, brood developmental time was shorter in the queenright compartment. These findings suggest that differences in body size are regulated by the brood interactions with the queen and workers, and not by factors inside the eggs that could vary along with colony development. Finally, we developed a model showing that the typical increase in worker number and the decrease in brood contact with the queen can account for the typical increase in body size. Similar self-organized social regulation of brood development may contribute to the optimization of growth and reproduction in additional social insects.     

Recognition of caste and mating status maintains monogyny in the ant Aphaenogaster senilis

In ants dispersing through colony fission, queens mate near their natal nest and found a new society with the help of workers. This allows potential future queens to challenge the mother queen’s reproductive monopoly. Conflicts might be resolved if the mated queen signals her presence and the workers control the developmental fate of the diploid larvae (whether they develop to worker or queen). In this study we sought to determine whether, in the fission-performing ant Aphaenogaster senilis, conflicts between queens for control of the colony are resolved by the resident queen signalling her mating status. Virgin queens were less effective than newly mated queens in inhibiting queen rearing. Moreover, potential challenger queens were recognized and heavily aggressed independent of mating status. Chemical analyses showed that mating status was associated with changes in cuticular hydrocarbon and poison gland composition, but not in Dufour’s gland composition. Cuticular dimethylalkanes were identified as potential constituents that signal both caste (present in queens only) and mating status (mated queens have higher amounts). We hypothesised that pheromone emission by virgin queens did not reach the threshold needed to fully inhibit larval development into queens but was sufficiently high to stimulate overt aggression by mated queens. These findings provide evidence for the complexity of chemical communication in social insects, in which a small number of signals may have a variety of effects, depending on the context.     

Demography, demand, death, and the seasonal allocation of labor in the Florida harvester ant (Pogonomyrmex badius)

As a self-organizing entity, an ant colony must divide a limited number of workers among numerous competing functions. Adaptive patterns of labor allocation should vary with colony need across each annual cycle, but remain almost entirely undescribed in ants. Allocation to foraging in 55 field colonies of the Florida harvester ant (Pogonomyrmex badius) followed a consistent annual pattern over 4 years. Foragers preceded larvae in spring and peaked during maximal larval production in summer (0.37). In spring, proportion foraging increased due to an increase in forager number and reduction in colony size, and in late summer, it decreased as colony size increased through new worker birth and a loss of ～3 % of foragers per day. The removal of 50 % of the forager population revealed that, at the expense of larval survival, colonies did not draw workers from other castes to fill labor gaps. To determine if labor allocation was age specific, whole colonies were marked with cuticle color-specific wire belts and released, and each cohort's time to first foraging was noted. Workers that eclosed in summer alongside sexual alates darkened quickly and became foragers at ～43 days of age, whereas autumn-born workers required 200 or more days to do so. Following colony reproduction, these long-lived individuals foraged alongside short-lived, summer-born sisters during the next calendar year. Therefore, the large-scale, predictable patterns of labor allocation in P. badius appear to be driven by bimodal worker development rate and age structure, rather than worker responsiveness to changes in colony demand.     

Do leaf-cutter ants Atta colombica obtain their magnetic sensors from soil?

How animals sense, process, and use magnetic information remains elusive. In insects, magnetic particles are candidates for a magnetic sensor. Recent studies suggest that the ant Pachycondyla marginata incorporates iron-containing particles from soil. We used leaf-cutter ants Atta colombica to test whether soil contact is necessary for developing a functional magnetic compass. A. colombica is the only invertebrate known to calculate a path-integrated home vector using a magnetic compass. Here, we show that A. colombica requires contact with soil to incorporate magnetic particles that can be used as a magnetic compass; yet, we also show that ants can biosynthesize magnetic particles. Workers from a soil-free colony ignored a 90° shift in the horizontal component of the geomagnetic field, yet oriented homeward despite the occlusion of any geocentric cues. In contrast, workers from a soil-exposed colony oriented to an intermediate direction between their true and subjective home in the shifted field. Homeward orientations under shifted fields suggest that ants calculated a path-integrated vector using proprioceptive information. Strikingly, ants from the soil-free colony also had magnetic particles; yet, as observed by ferromagnetic resonance, these particles differed from those in soil-exposed ants and were not associated with a magnetic compass sensitive to this experimental manipulation.     

Why are larvae of the social parasite wasp <Emphasis Type="Italic">Polistes sulcifer</Emphasis> not removed from the host nest?

A challenge for parasites is how to evade the sophisticated detection and rejection abilities of potential hosts. Many studies have shown how insect social parasites overcome host recognition systems and successfully enter host colonies. However, once a social parasite has successfully usurped an alien nest, its brood still face the challenge of avoiding host recognition. How immature stages of parasites fool the hosts has been little studied in social insects, though this has been deeply investigated in birds. We look at how larvae of the paper wasp obligate social parasite Polistes sulcifer fool their hosts. We focus on cuticular hydrocarbons (CHCs), which are keys for adult recognition, and use behavioral recognition assays. Parasite larvae might camouflage themselves either by underproducing CHCs (odorless hypothesis) or by acquiring a chemical profile that matches that of their hosts. GC/MS analyses show that parasite larvae do not have lower levels of CHCs and that their CHCs profile is similar to the host larval profile but shows a reduced colony specificity. Behavioral tests show that the hosts discriminate against alien conspecific larvae from different colonies but are more tolerant towards parasite larvae. Our results demonstrate that parasite larvae have evolved a host larval profile, which overcomes the host colony recognition system probably because of the lower proportion of branched compounds compared to host larvae. In some ways, this is a similar hypothesis to the odorless hypothesis, but it assumes that the parasite larvae are covered by a chemical blend that is not meaningful to the host.     

The costs and benefits of cooperation between the Australian lycaenid butterfly,Jalmenus evagoras,and its attendant ants

Summary The larvae and pupae of the Australian lycaenid butterfly, Jalmenus evagoras associate mutualistically with ants in the genus Iridomyrmex. Four ant exclusion experiments in three field sites demonstrated that predation and parasitism of J. evagoras are so intense that individuals deprived of their attendant ants are unlikely to survive. Larvae and pupae of J. evagoras aggregate, and the mean number of attendant ants per individual increases with larval age and decreases with group size. Field observations showed that young larvae could gain more attendant ants per individual by joining the average size group of about 4 larvae than by foraging alone. Aggregation behaviour is influenced by ant attendance: young larvae and pupating fifth instars aggregated significantly more often on plants with ants than on plants where ants had been excluded. In return for tending and protecting the larvae, ants were rewarded by food secretions that can amount to as much as 409 mg dry biomass from a single host plant containing 62 larvae and pupae of J. evagoras over a 24 h period. Larval development in the laboratory lasted approximately a month, and larvae that were tended by ants developed almost 5 days faster than larvae that were not tended. However, tended individuals, particularly females, pupated at a significantly lower weight than their untended counterparts, and the adults that eclosed from these pupae were also lighter and smaller. On average, pupae that were tended by ants lost 25% more weight than untended pupae, and in contrast with larvae, they took longer to eclose than pupae that were not tended. These experimental results are discussed in terms of costs and benefits of association for both partners, and of aggregation for the lycaenids.     

Chemical mimicry in the root aphid parasitoid Paralipsis eikoae Yasumatsu (Hymenoptera: Aphidiidae) of the aphid-attending ant Lasius sakagamii Yamauchi & Hayashida (Hymenoptera: Formicidae)

Summary. An aphidiid wasp, Paralipsis eikoae, was associated with both Lasius niger and L. sakagamii attending the wormwood root aphid Sappaphis piri. An L. sakagamii worker was observed carrying a winged female P. eikoae to its nest with its mandible, but it did not kill the wasp. Once accepted by the ants, the wasp often mounted and rubbed against the worker ants and sometimes teased them to regurgitate food to itself. No workers in the colony attacked the wasp. Conspecific foreign workers, however, viciously attacked the wasp when encountered. Gas chromatography-mass spectrometry analyses showed that the accepted wasp had complex cuticular hydrocarbons that were very similar to those of its host ants, whereas the winged wasps collected outside the ant nest showed only a series of n-alkanes. Additionally, the accepted wasp had a hydrocarbon profile closer to that of its host ants than to the conspecific foreign ants. We believe the wasp mimics ant cuticular hydrocarbons to integrate into the ant nest, acquiring the hydrocarbons by mounting and rubbing against the ants. In contrast, the cuticular hydrocarbons of the emerged wasp contained larval and pupal hydrocarbons of L. sakagamii that were also similar to those of L. niger. Both ant species rejected adult workers of the other species but accepted their larvae and pupae. We suggest that the emerged P. eikoae mimics the cuticular hydrocarbons of these Lasius larvae and pupae, which allows P. eikoae to be accepted by both L. sakagamii and L. niger. Received 11 March 1998; accepted 22 July 1998.     

Effects of the social environment on the survival and fungal resistance of ant brood

The phenotype of social animals can be influenced by genetic, maternal and environmental effects, which include social interactions during development. In social insects, the social environment and genetic origin of brood can each influence a whole suite of traits, from individual size to caste differentiation. Here, we investigate to which degree the social environment during development affects the survival and fungal resistance of ant brood of known maternal origin. We manipulated one component of the social environment, the worker/brood ratio, of brood originating from single queens of Formica selysi. We monitored the survival of brood and measured the head size and ability to resist the entomopathogenic fungus Beauveria bassiana of the resulting callow workers. The worker/brood ratio and origin of eggs affected the survival and maturation time of the brood and the size of the resulting callow workers. The survival of the callow workers varied greatly according to their origin, both in controls and when challenged with B. bassiana. However, there was no interaction between the fungal challenge and either the worker/brood ratio or origin of eggs, suggesting that these factors did not affect parasite resistance in the conditions tested. Overall, the social conditions during brood rearing and the origin of eggs had a strong impact on brood traits that are important for fitness. We detected a surprisingly large amount of variation among queens in the survival of their brood reared in standard queenless conditions, which calls for further studies on genetic, maternal and social effects influencing brood development in the social insects.     

The use of simulation modeling to evaluate the mechanisms responsible for the nutritional benefits of food-for-protection mutualisms

In some mutualisms, a plant or insect provides a food resource in exchange for protection from herbivores, competitors or predators. This food resource can benefit the consumer, but the relative importance of different mechanisms responsible for this benefit is unclear. We used a colony-level simulation model to test the relative importance of increased larval production, increased worker foraging and increased worker survival for colony growth of fire ants, Solenopsis invicta, that consume plant-based foods. Increased food for larvae had the largest effect on colony growth of S. invicta followed by decreased worker mortality. Increased foraging rate had a small effect in the simulation model but data from a small laboratory experiment and another published study suggest that plant-based foods have little or no effect on foraging rates of S. invicta. Colony growth steadily increased the longer plant-based food was available and colonies were most responsive to plant-based food in the early summer (i.e., June). Our results demonstrate that population level simulation modeling can be a useful tool for examining the ecology of mutualistic interactions and the mechanisms through which species interact.     

The role of per-capita productivity in the evolution of small colony sizes in ants

The evolution of colony size in social insects is influenced by both extrinsic and colony-intrinsic factors. An important intrinsic trait, per-capita productivity, often declines in larger colonies. This pattern, known as Michener’s paradox, can limit the growth of insect societies. In this study, we first describe this problem, survey its occurrence across different ant species, and present a case study of eight cavity-dwelling ants with very small colony sizes. In these species, colonies might never reach sizes at which per-capita productivity decreases. However, in six out of the eight focal species, per-capita productivity did decline with increasing size, in accordance with other studies on per-capita productivity in ants. Several mechanisms, such as resource availability or nest-site limitation, may explain the decrease in per-capita productivity with increases in colony size in our focal species. In these central-place foragers, the individual foraging mode is expected to lead to an increase in travel time as colonies grow. We suggest that polydomy, the concomitant occupation of several nest sites, could serve as a potential strategy to overcome this limitation. Indeed, for one species, we show that polydomy can help to circumvent the reduction in productivity with increasing colony size, suggesting that limited resource availability causes the observed decrease in per-capita productivity. Finally, we discuss the influence of other factors, such as the nesting ecology and colony homeostasis, on the evolution of colony size in these cavity-dwelling ants.     

Role of Propagule Size in the Success of Incipient Colonies of the Invasive Argentine Ant

Abstract: Factors that contribute to the successful establishment of invasive species are often poorly understood. Propagule size is considered a key determinant of establishment success, but experimental tests of its importance are rare. We used experimental colonies of the invasive Argentine ant (   Linepithema humile ) that differed both in worker and queen number to test how these attributes influence the survivorship and growth of incipient colonies. All propagules without workers experienced queen mortality, in contrast to only 6% of propagules with workers. In small propagules (10–1,000 workers), brood production increased with worker number but not queen number. In contrast, per capita measures of colony growth decreased with worker number over these colony sizes. In larger propagules ( 1,000–11,000 workers), brood production also increased with increasing worker number, but per capita brood production appeared independent of colony size. Our results suggest that queens need workers to establish successfully but that propagules with as few as 10 workers can grow quickly. Given the requirements for propagule success in Argentine ants, it is not surprising how easily they spread via human commerce.     

Quorum sensing, recruitment, and collective decision-making during colony emigration by the ant Leptothorax albipennis

When its nest is damaged, a colony of the ant Leptothorax albipennis skillfully emigrates to the best available new site. We investigated how this ability emerges from the behaviors used by ants to recruit nestmates to potential homes. We found that, in a given emigration, only one-third of the colony's workers ever recruit. At first, they summon fellow recruiters via tandem runs, in which a single follower is physically led all the way to the new site. They later switch to recruiting the passive majority of the colony via transports, in which nestmates are simply carried to the site. After this switch, tandem runs continue sporadically but now run in the opposite direction, leading recruiters back to the old nest. Recruitment accelerates with the start of transport, which proceeds at a rate 3 times greater than that of tandem runs. The recruitment switch is triggered by population increase at the new site, such that ants lead tandem runs when the site is relatively empty, but change to transport once a quorum of nestmates is present. A model shows that the quorum requirement can help a colony choose the best available site, even when few ants have the opportunity to compare sites directly, because recruiters to a given site launch the rapid transport of the bulk of the colony only if enough active ants have been "convinced" of the worth of the site. This exemplifies how insect societies can achieve adaptive colony-level behaviors from the decentralized interactions of relatively poorly informed insects, each combining her own limited direct information with indirect cues about the experience of her nestmates.