Wolbachia-infected pharaoh ant colonies have higher egg production, metabolic rate, and worker survival.

Wolbachia is a widespread maternally-transmitted endosymbiotic bacteria with diverse phenotypic effects on its insect hosts, ranging from parasitic to mutualistic. Wolbachia commonly infects social insects, where it faces unique challenges associated with its hosts' caste-based reproductive division of labor and colony living. Here we dissect the benefits and costs of Wolbachia infection on life-history traits of the invasive pharaoh ant, Monomorium pharaonis, which are relatively short-lived and show natural variation in Wolbachia infection status between colonies. We quantified effects of Wolbachia infection on the lifespan of queen and worker castes, the egg-laying rate of queens across queen lifespan, and the metabolic rates of whole colonies and colony members. Infected queens laid more eggs than uninfected queens but had similar metabolic rates and lifespans. Interestingly, infected workers outlived uninfected workers. At the colony level, infected colonies were more productive due to increased queen egg-laying rates and worker longevity, and infected colonies had higher metabolic rates during peak colony productivity. While some effects of infection, such as elevated colony-level metabolic rates may be detrimental in more stressful natural conditions, we did not find any costs of infection under relatively benign laboratory conditions. Overall, our study emphasizes that Wolbachia infection can have beneficial effects on ant colony growth and worker survival in at least some environments.

Dynamics of Information Flow and Task Allocation of Social Insect Colonies: Impacts of Spatial Interactions and Task Switching.

Models of social interaction dynamics have been powerful tools for understanding the efficiency of information spread and the robustness of task allocation in social insect colonies. How workers spatially distribute within the colony, or spatial heterogeneity degree (SHD), plays a vital role in contact dynamics, influencing information spread and task allocation. We used agent-based models to explore factors affecting spatial heterogeneity and information flow, including the number of task groups, variation in spatial arrangements, and levels of task switching, to study: (1) the impact of multiple task groups on SHD, contact dynamics, and information spread, and (2) the impact of task switching on SHD and contact dynamics. Both models show a strong linear relationship between the dynamics of SHD and contact dynamics, which exists for different initial conditions. The multiple-task-group model without task switching reveals the impacts of the number and spatial arrangements of task locations on information transmission. The task-switching model allows task-switching with a probability through contact between individuals. The model indicates that the task-switching mechanism enables a dynamical state of task-related spatial fidelity at the individual level. This spatial fidelity can assist the colony in redistributing their workforce, with consequent effects on the dynamics of spatial heterogeneity degree. The spatial fidelity of a task group is the proportion of workers who perform that task and have preferential walking styles toward their task location. Our analysis shows that the task switching rate between two tasks is an exponentially decreasing function of the spatial fidelity and contact rate. Higher spatial fidelity leads to more agents aggregating to task location, reducing contact between groups, thus making task switching more difficult. Our results provide important insights into the mechanisms that generate spatial heterogeneity and deepen our understanding of how spatial heterogeneity impacts task allocation, social interaction, and information spread.

Impacts of seasonality and parasitism on honey bee population dynamics.

The honeybee plays an extremely important role in ecosystem stability and diversity and in the production of bee pollinated crops. Honey bees and other pollinators are under threat from the combined effects of nutritional stress, parasitism, pesticides, and climate change that impact the timing, duration, and variability of seasonal events. To understand how parasitism and seasonality influence honey bee colonies separately and interactively, we developed a non-autonomous nonlinear honeybee-parasite interaction differential equation model that incorporates seasonality into the egg-laying rate of the queen. Our theoretical results show that parasitism negatively impacts the honey bee population either by decreasing colony size or destabilizing population dynamics through supercritical or subcritical Hopf-bifurcations depending on conditions. Our bifurcation analysis and simulations suggest that seasonality alone may have positive or negative impacts on the survival of honey bee colonies. More specifically, our study indicates that (1) the timing of the maximum egg-laying rate seems to determine when seasonality has positive or negative impacts; and (2) when the period of seasonality is large it can lead to the colony collapsing. Our study further suggests that the synergistic influences of parasitism and seasonality can lead to complicated dynamics that may positively and negatively impact the honey bee colony's survival. Our work partially uncovers the intrinsic effects of climate change and parasites, which potentially provide essential insights into how best to maintain or improve a honey bee colony's health.

A modeling framework for adaptive collective defense: crisis response in social-insect colonies.

Living systems, from cells to superorganismic insect colonies, have an organizational boundary between inside and outside and allocate resources to defend it. Whereas the micro-scale dynamics of cell walls can be difficult to study, the adaptive allocation of workers to defense in social-insect colonies is more conspicuous. This is particularly the case for Tetragonisca angustula stingless bees, which combine different defensive mechanisms found across other colonial animals: (1) morphological specialization (distinct soldiers (majors) are produced over weeks); (2) age-based polyethism (young majors transition to guarding tasks over days); and (3) task switching (small workers (minors) replace soldiers within minutes under crisis). To better understand how these timescales of reproduction, development, and behavior integrate to balance defensive demands with other colony needs, we developed a demographic Filippov ODE system to study the effect of these processes on task allocation and colony size. Our results show that colony size peaks at low proportions of majors, but colonies die if minors are too plastic or defensive demands are too high or if there is a high proportion of quickly developing majors. For fast maturation, increasing major production may decrease defenses. This model elucidates the demographic factors constraining collective defense regulation in social insects while also suggesting new explanations for variation in defensive allocation at smaller scales where the mechanisms underlying defensive processes are not easily observable. Moreover, our work helps to establish social insects as model organisms for understanding other systems where the transaction costs for component turnover are nontrivial, as in manufacturing systems and just-in-time supply chains.

Decoding alarm signal propagation of seed-harvester ants using automated movement tracking and supervised machine learning.

Alarm signal propagation through ant colonies provides an empirically tractable context for analysing information flow through a natural system, with useful insights for network dynamics in other social animals. Here, we develop a methodological approach to track alarm spread within a group of harvester ants, Pogonomyrmex californicus. We initially alarmed three ants and tracked subsequent signal transmission through the colony. Because there was no actual standing threat, the false alarm allowed us to assess amplification and adaptive damping of the collective alarm response. We trained a random forest regression model to quantify alarm behaviour of individual workers from multiple movement features. Our approach translates subjective categorical alarm scores into a reliable, continuous variable. We combined these assessments with automatically tracked proximity data to construct an alarm propagation network. This method enables analyses of spatio-temporal patterns in alarm signal propagation in a group of ants and provides an opportunity to integrate individual and collective alarm response. Using this system, alarm propagation can be manipulated and assessed to ask and answer a wide range of questions related to information and misinformation flow in social networks.

Social consequences of energetically costly nest construction in a facultatively social bee.

Social groups form when the costs of breeding independently exceed fitness costs imposed by group living. The costs of independent breeding can often be energetic, especially for animals performing expensive behaviours, such as nest construction. To test the hypothesis that nesting costs can drive sociality by disincentivizing independent nest founding, we measured the energetics of nest construction and inheritance in a facultatively social carpenter bee (Xylocopa sonorina Smith), which bores tunnel nests in wood. We measured metabolic rates of bees excavating wood and used computerized tomography images of nesting logs to measure excavation volumes. From these data, we demonstrate costly energetic investments in nest excavation of a minimum 4.3 kJ per offspring provisioned, an expense equivalent to nearly 7 h of flight. This high, potentially prohibitive cost of nest founding may explain why females compete for existing nests rather than constructing new ones, often leading to the formation of social groups. Further, we found that nest inheritors varied considerably in their investment in nest renovation, with costs ranging more than 12-fold (from 7.08 to 89.1 kJ energy), probably reflecting differences in inherited nest quality. On average, renovation costs were lower than estimated new nest construction costs, with some nests providing major savings. These results suggest that females may join social groups to avoid steep energetic costs, but that the benefits of this strategy are not experienced equally.

The active ingredients of a mitotoxic fungicide negatively affect pollen consumption and worker survival in laboratory-reared honey bees (Apis mellifera).

Recent observations of many sublethal effects of pesticides on pollinators have raised questions about whether standard short-term laboratory tests of pesticide effects on survival are sufficient for pollinator protection. The fungicide Pristine® and its active ingredients (25.2% boscalid, 12.8% pyraclostrobin) have been reported to have low acute toxicity to caged honey bee workers, but many sublethal effects at field-relevant doses have been reported and Pristine® was recently found to increase worker pollen consumption, reduce worker longevity and colony populations at field relevant concentrations (Fisher et al. 2021). To directly compare these whole-colony field results to more standard laboratory toxicology tests, the effects of Pristine®, at a range of field-relevant concentrations, were assessed on the survival and pollen consumption of honey bee workers 0-14 days of age. Also, to separate the effects of the inert and two active ingredients, bees were fed pollen containing boscalid, pyraclostrobin, or pyraclostrobin plus boscalid, at concentrations matching those in the Pristine® treatments. Pyraclostrobin significantly reduced pollen consumption across the duration of the experiment, and dose-dependently reduced pollen consumption on days 12-14. Pristine® and boscalid significantly reduced pollen feeding rate on days 12-14. Boscalid reduced survival in a dose-dependent manner. Consumption of Pristine® or pyraclostrobin plus boscalid did not affect survival, providing evidence against strong negative effects of the inert ingredients in Pristine® and against negative synergistic effects of boscalid and pyraclostrobin. The stronger toxic effects of Pristine® observed in field colonies compared to this laboratory test, and the opposite responses of pollen consumption in the laboratory and field to Pristine®, show that standard laboratory toxicology tests can fail to predict responses of pollinators to pesticides and to provide protection.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera).

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Dynamics of social interactions, in the flow of information and disease spreading in social insects colonies: Effects of environmental events and spatial heterogeneity.

The relationship between division of labor and individuals' spatial behavior in social insect colonies provides a useful context to study how social interactions influence the spreading of elements (which could be information, virus or food) across distributed agent systems. In social insect colonies, spatial heterogeneity associated with variations of individual task roles, affects social contacts, and thus the way in which agent moves through social contact networks. We used an Agent Based Model (ABM) to mimic three realistic scenarios of elements' transmission, such as information, food or pathogens, via physical contact in social insect colonies. Our model suggests that individuals within a specific task interact more with consequences that elements could potentially spread rapidly within that group, while elements spread slower between task groups. Our simulations show a strong linear relationship between the degree of spatial heterogeneity and social contact rates, and that the spreading dynamics of elements follow a modified nonlinear logistic growth model with varied transmission rates for different scenarios. Our work provides important insights on the dual-functionality of physical contacts. This dual-functionality is often driven via variations of individual spatial behavior, and can have both inhibiting and facilitating effects on elements' transmission rates depending on environment. The results from our proposed model not only provide important insights on mechanisms that generate spatial heterogeneity, but also deepen our understanding of how social insect colonies balance the benefit and cost of physical contacts on the elements' transmission under varied environmental conditions.

Ecological drivers and reproductive consequences of non-kin cooperation by ant queens.

The fitness consequences of joining a group are highly dependent on ecological context, especially for non-kin. To assess the relationships between cooperation and environment, we examined variation in colony reproductive success for a harvester ant species that nests either solitarily or with multiple, unrelated queens, a social strategy known as primary polygyny. We measured the reproductive investment of colonies of solitary versus social nesting types at two sites, one with primarily single-queen colonies, and the other with a majority of polygynous nests. Our results were consistent with the hypothesis that cooperative nesting by unrelated ant queens is likely a selection response to difficult environments, rather than a strategy to maximize reproduction under favorable conditions. Fewer colonies at the primarily polygynous site reproduced than at the site with primarily single queen nests, and those that did had lower reproductive investment, as measured by number and total mass of reproductives. Assessment of ecological conditions also support the harsh environment hypothesis. Colony density in the multi-queen population was higher, and nearest neighbor distances were lower for non-reproducing than reproducing colonies. To more directly test the hypothesis that colony reproduction was ecologically constrained, we experimentally supplemented food resources for a subset of colonies at the primary polygyny site. Supplemented colonies increased reproductive investment levels to equal that of colonies at the single-queen population, further indicating that environmental pressures are severe where primary polygyny is dominant, and may drive the evolution of non-kin cooperation in this context.

Differentiating causality and correlation in allometric scaling: ant colony size drives metabolic hypometry.

Metabolic rates of individual animals and social insect colonies generally scale hypometrically, with mass-specific metabolic rates decreasing with increasing size. Although this allometry has wide ranging effects on social behaviour, ecology and evolution, its causes remain controversial. Because it is difficult to experimentally manipulate body size of organisms, most studies of metabolic scaling depend on correlative data, limiting their ability to determine causation. To overcome this limitation, we experimentally reduced the size of harvester ant colonies (Pogonomyrmex californicus) and quantified the consequent increase in mass-specific metabolic rates. Our results clearly demonstrate a causal relationship between colony size and hypometric changes in metabolic rate that could not be explained by changes in physical density. These findings provide evidence against prominent models arguing that the hypometric scaling of metabolic rate is primarily driven by constraints on resource delivery or surface area/volume ratios, because colonies were provided with excess food and colony size does not affect individual oxygen or nutrient transport. We found that larger colonies had lower median walking speeds and relatively more stationary ants and including walking speed as a variable in the mass-scaling allometry greatly reduced the amount of residual variation in the model, reinforcing the role of behaviour in metabolic allometry. Following the experimental size reduction, however, the proportion of stationary ants increased, demonstrating that variation in locomotory activity cannot solely explain hypometric scaling of metabolic rates in these colonies. Based on prior studies of this species, the increase in metabolic rate in size-reduced colonies could be due to increased anabolic processes associated with brood care and colony growth.

Gene expression and variation in social aggression by queens of the harvester ant Pogonomyrmex californicus.

A key requirement for social cooperation is the mitigation and/or social regulation of aggression towards other group members. Populations of the harvester ant Pogonomyrmex californicus show the alternate social phenotypes of queens founding nests alone (haplometrosis) or in groups of unrelated yet cooperative individuals (pleometrosis). Pleometrotic queens display an associated reduction in aggression. To understand the proximate drivers behind this variation, we placed foundresses of the two populations into social environments with queens from the same or the alternate population, and measured their behaviour and head gene expression profiles. A proportion of queens from both populations behaved aggressively, but haplometrotic queens were significantly more likely to perform aggressive acts, and conflict escalated more frequently in pairs of haplometrotic queens. Whole-head RNA sequencing revealed variation in gene expression patterns, with the two populations showing moderate differentiation in overall transcriptional profile, suggesting that genetic differences underlie the two founding strategies. The largest detected difference, however, was associated with aggression, regardless of queen founding type. Several modules of coregulated genes, involved in metabolism, immune system and neuronal function, were found to be upregulated in highly aggressive queens. Conversely, nonaggressive queens exhibited a striking pattern of upregulation in chemosensory genes. Our results highlight that the social phenotypes of cooperative vs. solitary nest founding tap into a set of gene regulatory networks that seem to govern aggression level. We also present a number of highly connected hub genes associated with aggression, providing opportunity to further study the genetic underpinnings of social conflict and tolerance.

Mechanistic modeling of alarm signaling in seed-harvester ants.

Ant colonies demonstrate a finely tuned alarm response to potential threats, offering a uniquely manageable empirical setting for exploring adaptive information diffusion within groups. To effectively address potential dangers, a social group must swiftly communicate the threat throughout the collective while conserving energy in the event that the threat is unfounded. Through a combination of modeling, simulation, and empirical observations of alarm spread and damping patterns, we identified the behavioral rules governing this adaptive response. Experimental trials involving alarmed ant workers (Pogonomyrmex californicus) released into a tranquil group of nestmates revealed a consistent pattern of rapid alarm propagation followed by a comparatively extended decay period [1]. The experiments in [1] showed that individual ants exhibiting alarm behavior increased their movement speed, with variations in response to alarm stimuli, particularly during the peak of the reaction. We used the data in [1] to investigate whether these observed characteristics alone could account for the swift mobility increase and gradual decay of alarm excitement. Our self-propelled particle model incorporated a switch-like mechanism for ants' response to alarm signals and individual variations in the intensity of speed increased after encountering these signals. This study aligned with the established hypothesis that individual ants possess cognitive abilities to process and disseminate information, contributing to collective cognition within the colony (see [2] and the references therein). The elements examined in this research support this hypothesis by reproducing statistical features of the empirical speed distribution across various parameter values.

Body mass and cuticular hydrocarbon profiles, but not queen number, underlie worker desiccation resistance in a facultatively polygynous harvester ant (Pogonomyrmex californicus).

As small-bodied terrestrial organisms, insects face severe desiccation risks in arid environments, and these risks are increasing under climate change. Here, we investigate the physiological, chemical, and behavioral mechanisms by which harvester ants, one of the most abundant arid-adapted insect groups, cope with desiccating environmental conditions. We aimed to understand how body size, cuticular hydrocarbon profiles, and queen number impact worker desiccation resistance in the facultatively polygynous harvester ant, Pogonomyrmex californicus. We measured survival at 0% humidity of field-collected worker ants sourced from three closely situated populations within a semi-arid region of southern California. These populations vary in queen number, with one population dominated by multi-queen colonies (primary polygyny), one population dominated by single-queen colonies, and one containing an even mix of single- and multi-queen colonies. We found no effect of population on worker survival in desiccation assays, suggesting that queen number does not influence colony desiccation resistance. Across populations, however, body mass and cuticular hydrocarbon profiles significantly predicted desiccation resistance. Larger-bodied workers survived longer in desiccation assays, emphasizing the importance of reduced surface area-to-volume ratios in maintaining water balance. Additionally, we observed a positive relationship between desiccation resistance and the abundance of n-alkanes, supporting previous work that has linked these high-melting point compounds to improved body water conservation. Together, these results contribute to an emerging model explaining the physiological mechanisms of desiccation resistance in insects.

Developmental evolution in social insects: regulatory networks from genes to societies.

The evolution and development of complex phenotypes in social insect colonies, such as queen-worker dimorphism or division of labor, can, in our opinion, only be fully understood within an expanded mechanistic framework of Developmental Evolution. Conversely, social insects offer a fertile research area in which fundamental questions of Developmental Evolution can be addressed empirically. We review the concept of gene regulatory networks (GRNs) that aims to fully describe the battery of interacting genomic modules that are differentially expressed during the development of individual organisms. We discuss how distinct types of network models have been used to study different levels of biological organization in social insects, from GRNs to social networks. We propose that these hierarchical networks spanning different organizational levels from genes to societies should be integrated and incorporated into full GRN models to elucidate the evolutionary and developmental mechanisms underlying social insect phenotypes. Finally, we discuss prospects and approaches to achieve such an integration.

Soldier neural architecture is temporarily modality specialized but poorly predicted by repertoire size in the stingless bee Tetragonisca angustula.

Individual heterogeneity within societies provides opportunities to test hypotheses about adaptive neural investment in the context of group cooperation. Here, we explore neural investment in defense specialist soldiers of the eusocial stingless bee (Tetragonisca angustula) which are age subspecialized on distinct defense tasks and have an overall higher lifetime task repertoire than other sterile workers within the colony. Consistent with predicted behavioral demands, soldiers had higher relative visual (optic lobe) investment than nonsoldiers but only during the period when they were performing the most visually demanding defense task (hovering guarding). As soldiers aged into the less visually demanding task of standing guarding this difference disappeared. Neural investment was otherwise similar across all colony members. Despite having larger task repertoires, soldiers had similar absolute brain size and the smaller relative brain size compared to other workers, meaning that lifetime task repertoire size was a poor predictor of brain size. Both high behavioral specialization in stable environmental conditions and reassignment across task groups during a crisis occur in T. angustula. The differences in neurobiology we report here are consistent with these specialized but flexible defense strategies. This work broadens our understanding of how neurobiology mediates age and morphological task specialization in highly cooperative societies.

Social Factors in Heat Survival: Multiqueen Desert Ant Colonies Have Higher and More Uniform Heat Tolerance.

AbstractInvestigations of thermally adaptive behavioral phenotypes are critical for both understanding climate as a selective force and predicting global species distributions under climate change conditions. Cooperative nest founding is a common strategy in harsh environments for many species and can enhance growth and competitive advantage, but whether this social strategy has direct effects on thermal tolerance was previously unknown. We examined the effects of alternative social strategies on thermal tolerance in a facultatively polygynous (multiqueen) desert ant, Pogonomyrmex californicus, asking whether and how queen number affects worker thermal tolerances. We established and reared lab colonies with one to four queens, then quantified all colony member heat tolerances (maximum critical temperature [CTmax]). Workers from colonies with more queens had higher and less variant CTmax. Our findings resemble weak link patterns, in which colony group thermal performance is improved by reducing frequencies of the most temperature-vulnerable individuals. Using ambient temperatures from our collection site, we show that multiqueen colonies have thermal tolerance distributions that enable increased midday foraging in hot desert environments. Our results suggest advantages to polygyny under climate change scenarios and raise the question of whether improved thermal tolerance is a factor that has enabled the success of polygyne species in other climatically extreme environments.

Seasonal variability in physiology and behavior affect the impact of fungicide exposure on honey bee (Apis mellifera) health.

Honey bee pollination services are of tremendous agricultural and economic importance. Despite this, honey bees and other pollinators face ongoing perils, including population declines due to a variety of environmental stressors. Fungicides may be particularly insidious stressors for pollinators due to their environmental ubiquity and widespread approval for application during crop bloom. The mechanisms by which fungicides affect honey bees are poorly understood and any seasonal variations in their impact are unknown. Here we assess the effects on honey bee colonies of four-week exposure (the approximate duration of the almond pollination season) of a fungicide, Pristine® (25.2% boscalid, 12.8% pyraclostrobin), that has been commonly used for almonds. We exposed colonies to Pristine® in pollen patties placed into the hive, in either summer or fall, and assessed colony brood and worker populations, colony pollen collection and consumption, and worker age of first foraging and longevity. During the summer, Pristine® exposure induced precocious foraging, and reduced worker longevity resulting in smaller colonies. During the fall, Pristine® exposure induced precocious foraging but otherwise had no significant measured effects. During the fall, adult and brood population levels, and pollen consumption and collection, were all much lower, likely due to preparations for winter. Fungicides and other pesticides may often have reduced effects on honey bees during seasons of suppressed colony growth due to bees consuming less pollen and pesticide.

Mathematical modeling on obligate mutualism: Interactions between leaf-cutter ants and their fungus garden.

We propose a simple mathematical model by applying Michaelis-Menton equations of enzyme kinetics to study the mutualistic interaction between the leaf cutter ant and its fungus garden at the early stage of colony expansion. We derive sufficient conditions on the extinction and coexistence of these two species. In addition, we give a region of initial condition that leads to the extinction of two species when the model has an interior attractor. Our global analysis indicates that the division of labor by worker ants and initial conditions are two important factors that determine whether leaf cutter ants' colonies and their fungus garden can survive and grow or not. We validate the model by comparing model simulations and data on fungal and ant colony growth rates under laboratory conditions. We perform sensitive analysis of the model based on the experimental data to gain more biological insights on ecological interactions between leaf-cutter ants and their fungus garden. Finally, we give conclusions and discuss potential future work.