Operant conditioning of antennal muscle activity in the honey bee (Apis mellifera L.)

Antennal movements of the honey bee can be conditioned operantly under laboratory conditions. Using this behavioural paradigm we have developed a preparation in which the activity of a single antennal muscle has been operantly conditioned. This muscle, the fast flagellum flexor muscle, is innervated by an identified motoneuron whose action potentials correlate 1:1 with the muscle potentials. The activity of the fast flagellum flexor muscle was recorded extracellularly from the scapus of the antenna. The animal was rewarded with a drop of sucrose solution whenever the muscle activity exceeded a defined reward threshold. The reward threshold was one standard deviation above the mean spontaneous frequency prior to conditioning. After ten conditioning trials, the frequency of the muscle potentials had increased significantly compared to the spontaneous frequency. The conditioned changes of frequency were observed for 30 min after conditioning. No significant changes of the frequency were found in the yoke control group. The firing pattern of the muscle potentials did not change significantly after conditioning or feeding. Fixing the antennal joints reduces or abolishes associative operant conditioning. The conditioned changes of the frequency of muscle potentials in the freely moving antenna are directly comparable to the behavioural changes during operant conditioning. Accepted: 29 March 2000     

Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning

Although the forelegs of honeybees are one of their main gustatory appendages, tarsal gustation in bees has never been systematically studied. To provide a more extensive account on honeybee tarsal gustation, we performed a series of behavioral experiments aimed at characterizing (1) tarsal sucrose sensitivity under different experimental conditions and (2) the capacity of tarsal sucrose stimulation to support olfactory conditioning. We quantified the proboscis extension reflex to tarsal sucrose stimulation and to odors paired with tarsal sucrose stimulation, respectively. Our experiments show that tarsal sucrose sensitivity is lower than antennal sucrose sensitivity and can be increased by starvation time. In contrast, antennae amputation decreases tarsal sucrose sensitivity. Furthermore, we show that tarsal sucrose stimulation can support olfactory learning and memory even if the acquisition level reached is relatively low (40%).     

Function and central projections of gustatory receptor neurons on the antenna of the noctuid moth Spodoptera littoralis

Chemosensory information is crucial for most insects to feed and reproduce. Olfactory signals are mainly used at a distance, whereas gustatory stimuli play an important role when insects directly contact chemical substrates. In noctuid moths, although the antennae are the main olfactory organ, they also bear taste sensilla. These taste sensilla detect sugars and hence are involved in appetitive learning but could also play an important role in food evaluation by detecting salts and bitter substances. To investigate this, we measured the responses of individual taste sensilla on the antennae of Spodoptera littoralis to sugars and salts using tip recordings. We also traced the projections of their neuronal axons into the brain. In each sensillum, we found one or two neurons responding to sugars: one NaCl-responsive and one water-sensitive neuron. Responses of these neurons were dose-dependent and similar across different locations on the antenna. Responses were dependent on the sex for sucrose and on both sex and location for glucose and fructose. We did not observe a spatial map for the projections from specific regions of the antennae to the deutocerebrum or the tritocerebrum/suboesophageal ganglion complex. In accordance with physiological recordings, back-fills from individual sensilla revealed up to four axons, in most cases targeting different projection zones.     

Antennal movements induced by odour and central projection of the antennal neurones in the honey-bee

Movements of the antennae induced by odour were investigated. Odour presented to the antenna of one side induced both antennae to move to that side. The EMGs recorded from the flexor muscles of both scapes showed that the latency of the movement of the ipsilateral flagellum when induced by odour was about 71 msec shorter than that of the contralateral flagellum. Recording electrical activities from the antennal nerve showed that there are more than 14 neurones in the antenno-motor externus.The distribution of the antennal nerve in the brain was investigated histologically by the injection of fluorescent dye. Antennal sensory neurones terminated at the glomeruli in the antennal lobe, in the dorsal lobe, in the protocerebrum, and in the commissural part of the suboesophageal ganglion. Injection of the fluorescent dye into the antennal nerve after degeneration of the antennal sensory neurones showed that the antennal motoneurones run in the ventral side of the antennal and dorsal lobes, and terminate in the marginal region of the ipsilateral oesophageal connective.The difference in latency of odour-induced flagellar movements is discussed in relation to the histological results and the unitary responses in the brain reported previously.     

The functions of antennal mechanoreceptors and antennal joints in tactile discrimination of the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Honeybees learn and discriminate excellently between different surface structures and different forms of objects, which they scan with their antennae. The sensory plate on the antennal tip plays a key role in the perception of mechanosensory and gustatory information. It is densely covered with small tactile hairs and carries a few large taste hairs. Both types of sensilla contain a mechanoreceptor, which is involved in the antennal scanning of an object. Our experiments test the roles of the mechanoreceptors on the antennal tip in tactile antennal learning and discrimination. Joints between head capsule and scapus and between scapus and pedicellus enable the bee to perform three-dimensional movements when they scan an object. The role of these joints in tactile antennal learning and discrimination is studied in separate experiments. The mechanoreceptors on the antennal tip were decisive for surface discrimination, but not for tactile acquisition or discrimination of shapes. When the scapus–pedicellus joint or the headcapsule–scapus joint was fixed on both antennae, tactile learning was still apparent but surface discrimination was abolished. Fixing both scapi to the head capsule reduced tactile acquisition.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

The antennal motor system of the stick insect Carausius morosus: anatomy and antennal movement pattern during walking

The stick insect Carausius morosus continuously moves its antennae during locomotion. Active antennal movements may reflect employment of antennae as tactile probes. Therefore, this study treats two basic aspects of the antennal motor system: First, the anatomy of antennal joints, muscles, nerves and motoneurons is described and discussed in comparison with other species. Second, the typical movement pattern of the antennae is analysed, and its spatio-temporal coordination with leg movements described. Each antenna is moved by two single-axis hinge joints. The proximal head-scape joint is controlled by two levator muscles and a three-partite depressor muscle. The distal scape-pedicel joint is controlled by an antagonistic abductor/ adductor pair. Three nerves innervate the antennal musculature, containing axons of 14-17 motoneurons, including one common inhibitor. During walking, the pattern of antennal movement is rhythmic and spatiotemporally coupled with leg movements. The antennal abduction/adduction cycle leads the protraction/retraction cycle of the ipsilateral front leg with a stable phase shift. During one abduction/adduction cycle there are typically two levation/depression cycles, however, with less strict temporal coupling than the horizontal component. Predictions of antennal contacts with square obstacles to occur before leg contacts match behavioural performance, indicating a potential role of active antennal movements in obstacle detection.     

Tactile motor learning in the antennal system of the honeybee (Apis mellifera L.)

Honeybees fixed in small tubes scan an object within the range of the antennae by touching it briefly and frequently. In our experiments the animals were able to scan an object for several minutes with the antennae. After moving the object out of the range of the antennae, the animals showed antennal movements for several minutes that were correlated with the position of the removed object. These changes of antennal movements are called “behavioural plasticity” and are interpreted as a form of motor learning. Bees showed behavioural plasticity only for objects with relatively large surfaces. Plasticity was more pronounced in bees whose compound eyes were occluded. Behavioural plasticity was related to the duration of object presentation. Repeated presentations of the object increased the degree of plasticity. After presentation durations of 30 min the animals showed a significant increase of antennal positions related to the surface of the object and avoidance of areas corresponding to the edges. Behavioural plasticity was compared with reward-dependent learning by conditioning bees to objects. The results of motor learning and reward-dependent conditioning suggest that bees have tactile spatial memory. Accepted: 13 May 1997     

Tactile localisation: the function of active antennal movements in the crayfishCherax destructor

Video recordings and single frame analysis were used to study the function of the second antennae of crayfish (Cherax destructor) as a sensory system in freely behaving animals. Walking crayfish move their antennae back and forth through horizontal angles of 100 degrees and more, relative to the body long axis. At rest, animals tend to hold their antennae at angular positions between 20 and 50 degrees. Movements of the two antennae are largely independent of each other. Before and during a turn of the body the ipsilateral antenna is moved into the direction of the turn. Solid objects are explored by repeatedly moving the antennae towards and across them. Both seeing and blinded crayfish can locate stationary objects following antennal contact. On antennal contact with a small novel object, a moving animal withdraws its antenna and attacks the object. When the antenna of a blinded crayfish is lightly touched with a brush the animal turns and attacks the point of stimulation. The direction taken and the distance covered during an attack can be correlated with: the angle at which the antenna is held at the moment of contact and the distance along the antennal flagellum at which the stimulus is applied. From behavioural evidence we conclude that crayfish use information about the angular position of their antennae and about the position of stimulated mechanoreceptors along the antennal flagellum to locate objects in their environment. We suggest ways in which an active tactile system-like the crayfish's antennae--could supply animals with information about the three-dimensional layout of their environment.     

Identification of serotonin- and vasopressin immunoreactivities in the suprachiasmatic nucleus of four mammalian species

Summary Cobalt fills from small, defined regions of the antenna in D. melanogaster show that the three types of sensilla on the third segment, the flagellum, and a fourth sensillum located in the arista, project into the glomeruli of the antennal lobe. We have identified 19 glomeruli in each lobe, according to their location, shape, and size. At least ten of these represent major projection areas of flagellar or aristal sensilla. The large majority of glomeruli is innervated from both antennae, but a small group of five receive exclusively ipsilateral input. A particular sensory fiber appears to terminate only in one specific glomerulus, either in the ipsilateral or in both lobes. Fills from flagellar regions bearing a single type of sensillum, yield a specific pattern of glomeruli containing stained terminals. Aristal projections remain strictly ipsilateral, whereas those from the other sensilla consist of an ipsilateral and a bilateral component. When filling from different points in an area bearing one type of sensillum, similar projections are produced, suggesting that projection patterns observed reflect predominantly the type of sensillum rather than its location on the flagellum. Accordingly, individual glomeruli might represent functional units, each receiving antennal input in a characteristic combination.We are indebted to Dr. H. Tobler for critical comments. R.F.S. was supported by the Swiss National Foundation (Grant No. 3.541-0.79) as well as a Travel Aid by the Swiss Academy of Sciences     

Gustatory feedback affects feeding related motor pattern generation in starved 3rd instar larvae of Calliphora vicina

Gustatory feedback allows animals to distinguish between edible and noxious food and adapts centrally generated feeding motor patterns to environmental demands. In reduced preparations obtained from starved Calliphora larvae, putatively appetitive (ethanol), aversive (sodium acetate) and neutral (glucose) gustatory stimuli were applied to the anterior sense organs. The resulting sensory response was recorded from the maxillary- and antennal nerves. All three stimuli increased the neural activity in both nerves. Recordings obtained from the antennal nerve to monitor the activation pattern of the cibarial dilator muscles, demonstrated an effect of gustatory input on the central pattern generator for feeding. Ethanol consistently enhanced the rhythmic activity of the CDM motor neurons either by speeding up the rhythm or by increasing the burst duration. Ethanol also had an enhancing effect on the motor patterns of a protractor muscle which moves the cephalopharyngeal skeleton relative to the body. Sodium acetate showed a state dependent effect: in preparations without spontaneous CDM activity it initiated rhythmic motor patterns, while an ongoing CDM rhythm was inhibited. Surprisingly glucose had an enhancing effect which was less pronounced than that of ethanol. Gustatory feedback therefore can modify and adapt the motor output of the multifunctional central pattern generator for feeding.     

Processing of antennal information in extrinsic mushroom body neurons of the bee brain

Summary In the bee brain neural activity of interneurons of the inner antenno-cerebral tract (inputs to the mushroom body) and extrinsic neurons of the-lobe (output cells) was recorded intracellularly. The cells were stained with Lucifer Yellow. The response characteristics of the neurons to light, various antennal stimuli and mechanical stimuli to thorax and abdomen were studied.The cells of the inner antenno-cerebral tract (ACT) have uniglomerular dendritic arborizations in the antennal lobe and send projections into the calyces of the ipsilateral mushroom body and the lateral protocerebral lobe. 93% of the neurons are bi- or multimodal. No responses to light stimuli were found. Tactile stimuli to the antennae are only effective when applied ipsilaterally. Only one neuron showed marked differences in the responses to the qualitative testing of three odors: rose, lavender and isoamyl acetate.The cells can be classified according to their response characteristics; the following response types were found: (1) inhibitory responses to the stimuli, (2) inhibitory responses to olfactory and excitatory responses to mechanical stimuli or vice versa, (3) excitatory responses to mechanical and sugar water stimuli, (4) excitation to olfactory stimuli and to touching the antenna with a drop of water or sugar water, (5) excitation to mechanical stimuli to head, thorax and abdomen and inhibition to sugar water stimuli.The recorded extrinsic-lobe neurons have small dendritic bands perpendicular to the Kenyon cells, their axons project to the contralateral median protocerebrum. These cells have ipsilateral antennal and mostly ipsilateral optic inputs and process information from thoracic and abdominal mechanoreceptors. All responses are excitatory.The recordings suggest that the mushroom bodies are multimodal integration centers, where antennal information is first combined with visual inputs.Abbreviation ACT antenno-cerebral tract     

Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology

Physiological mechanisms of antennal sucrose perception in the honey bee were analysed using behavioural and electrophysiological methods. Following sucrose stimulation of the tip of a freely moving antenna, the latency of proboscis extension was 320–340 ms, 80–100 ms after the first activity in muscle M17 controlling this response. When bees were allowed to actively touch a sucrose droplet with one antenna, contacts with the solution were frequent with durations of 10–20 ms and average intervals between contacts of approximately 40 ms. High sucrose concentrations led to short and frequent contacts. The proboscis response and M17 activity were largely independent of stimulus duration and temporal pattern. Taste hairs of the antennal tip displayed spike responses to sucrose concentrations down to at least 0.1%. The first 25 ms of the response were suitable for discrimination of sucrose concentrations. This time interval corresponds to the duration of naturally occurring gustatory stimuli. Sucrose responses between different hairs on the same antenna showed a high degree of variability, ranging from less than five to over 40 spikes per 0.5 s for a stimulus of 0.1% sucrose. This variability of receptor responses extends the dynamic range of sucrose perception over a large range of concentrations.     

六斑月瓢虫触角及感觉器扫描电镜观察

[目的] 明确六斑月瓢虫雌雄成虫触角感觉器种类、分布及形态特征。[方法] 利用扫描电子显微镜对六斑月瓢虫雌、雄成虫触角形态及触角感受器超微结构进行观察。[结果] 六斑月瓢虫成虫触角由柄节、梗节和鞭节组成,柄节长度与宽度显著大于梗节长度与宽度;鞭节分为9个亚节,末端3节横向膨大呈锤状。雌雄成虫触角上共有8种感觉器:刺形感觉器(SC)、毛形感觉器(ST)、锥形感觉器(SB)、腔形感觉器(CaS)、钟形感觉器(CS)、哑铃形感觉器(DS)、香肠形感觉器(SS)及B?hm氏鬃毛感觉器(BB)。以毛形感觉器和刺形感觉器分布最广,遍布触角;B?hm氏鬃毛仅存在于触角柄节与梗节;触角鞭节第9亚节顶端密布7种触角感觉器。六斑月瓢虫雌雄成虫触角长度、触角感觉器类型及分布无显著差异。[结论] 六斑月瓢虫成虫触角上共有8种感觉器,其触角可能具有感知机械刺激、识别化学信息素及感受温湿度变化的作用。本研究为进一步了解六斑月瓢虫触角与其行为间的关系提供基础资料。     

Descending influences from the lateral hypothalamus and amygdala converge onto medullary taste neurons

The lateral hypothalamus (LH) and the central nucleus of the amygdala (CeA) exert an influence on many aspects of ingestive behavior. These nuclei receive projections from several areas carrying gustatory and viscerosensory information, and send axons to these nuclei as well, including the nucleus of the solitary tract (NST). Gustatory responses of NST neurons are modulated by stimulation of the LH and the CeA, and by several physiological factors related to ingestive behavior. We investigated the effect of both LH and CeA stimulation on the activity of 215 taste-responsive neurons in the hamster NST. More than half of these neurons (113/215) were modulated by electrical stimulation of the LH and/or CeA; of these, 52 cells were influenced by both areas, often bilaterally. The LH influenced more neurons than the CeA (101 versus 64 cells). Contralateral stimulation of these forebrain areas was more often effective (144 responses) than ipsilateral (74). Modulatory effects were mostly excitatory (102 cells); 11 cells were inhibited, mostly by ipsilateral LH stimulation. A subset of these cells (n = 25) was examined for the effects of microinjection of DL-homocysteic acid (DLH), a glutamate receptor agonist, into the LH and/or CeA. The effects of electrical stimulation were completely mimicked by DLH, indicating that cell somata in and around the stimulating sites were responsible for these effects. Other cells (n = 25) were tested for the effects of electrical stimulation of the LH and/or CeA on the responses to taste stimulation of the tongue (32 mM sucrose, NaCl and quinine hydrochloride, and 3.2 mM citric acid). Responses to taste stimuli were enhanced by the excitatory influence of the LH and/or CeA. These data demonstrate that descending influences from the LH and CeA reach many of the same cells in the gustatory NST and can modulate their responses to taste stimulation.     

Reception and learning of electric fields in bees

Honeybees, like other insects, accumulate electric charge in flight, and when their body parts are moved or rubbed together. We report that bees emit constant and modulated electric fields when flying, landing, walking and during the waggle dance. The electric fields emitted by dancing bees consist of low- and high-frequency components. Both components induce passive antennal movements in stationary bees according to Coulomb''s law. Bees learn both the constant and the modulated electric field components in the context of appetitive proboscis extension response conditioning. Using this paradigm, we identify mechanoreceptors in both joints of the antennae as sensors. Other mechanoreceptors on the bee body are potentially involved but are less sensitive. Using laser vibrometry, we show that the electrically charged flagellum is moved by constant and modulated electric fields and more strongly so if sound and electric fields interact. Recordings from axons of the Johnston organ document its sensitivity to electric field stimuli. Our analyses identify electric fields emanating from the surface charge of bees as stimuli for mechanoreceptors, and as biologically relevant stimuli, which may play a role in social communication.     

Distribution of chemo- and mechanoreceptors on the antennae and maxillae of Busseola fusca larvae

The stem borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is a major pest of maize, Zea mays L., and sorghum, Sorghum bicolor (L.) Moench (both Poaceae), in sub-Saharan Africa. Like in many other lepidopteran insects, the success of B. fusca in recognizing and colonizing a limited variety of plants is based on the interaction between its sensory systems and the physicochemical characteristics of its immediate environment. The sensilla on the maxillary galeae of B. fusca larvae are typical of Lepidoptera and comprise two uniporous styloconic sensilla, which are contact chemoreceptors, three basiconic sensilla, and two aporous sensilla chaetica. The maxillary palp is two-segmented and has eight small basiconic sensilla at the tip, which were also found to be gustatory. The antennae of B. fusca larvae are short and simple. The sensilla of the antenna are composed of two aporous sensilla chaetica, three multiporous cone-shaped basiconic sensilla, three small basiconic sensilla, and one aporous styloconic sensillum. The basiconic sensillum located on the third antennal segment displayed a contact chemoreception response. The other basiconic sensilla did not show any action potential activity in tip-recording tests. The significant and positive dose–response curve obtained for the antennal basiconic sensillum with sucrose indicated for the first time the presence of gustatory chemoreceptors on the antennae of a lepidopteran larva.     

Brief exposure to sensory cues elicits stimulus-nonspecific general sensitization in an insect

The effect of repeated exposure to sensory stimuli, with or without reward is well known to induce stimulus-specific modifications of behaviour, described as different forms of learning. In recent studies we showed that a brief single pre-exposure to the female-produced sex pheromone or even a predator sound can increase the behavioural and central nervous responses to this pheromone in males of the noctuid moth Spodoptera littoralis. To investigate if this increase in sensitivity might be restricted to the pheromone system or is a form of general sensitization, we studied here if a brief pre-exposure to stimuli of different modalities can reciprocally change behavioural and physiological responses to olfactory and gustatory stimuli. Olfactory and gustatory pre-exposure and subsequent behavioural tests were carried out to reveal possible intra- and cross-modal effects. Attraction to pheromone, monitored with a locomotion compensator, increased after exposure to olfactory and gustatory stimuli. Behavioural responses to sucrose, investigated using the proboscis extension reflex, increased equally after pre-exposure to olfactory and gustatory cues. Pheromone-specific neurons in the brain and antennal gustatory neurons did, however, not change their sensitivity after sucrose exposure. The observed intra- and reciprocal cross-modal effects of pre-exposure may represent a new form of stimulus-nonspecific general sensitization originating from modifications at higher sensory processing levels.     

The antennal system and cockroach evasive behavior. I. Roles for visual and mechanosensory cues in the response

Cockroaches escape from predators by turning and then running. This behavior can be elicited when stimuli deflect one of the rostrally located and highly mobile antennae. We analyzed the behavior of cockroaches, under free-ranging conditions with videography or tethered in a motion tracking system, to determine (1) how antennal positional dynamics influence escape turning, and (2) if visual cues have any influence on antennal mediated escape. The spatial orientation of the long antennal flagellum at the time of tactile stimulation affected the direction of resultant escape turns. However, the sign of flagellar displacement caused by touch stimuli, whether it was deflected medially or laterally for example, did not affect the directionality of turns. Responsiveness to touch stimuli, and escape turn performance, were not altered by blocking vision. However, because cockroaches first orient an antenna toward stimuli entering the peripheral visual field, turn direction can be indirectly influenced by visual input. Finally, when vision was blocked, the run phase of escape responses displayed reduced average velocities and distances traveled. Our results suggest that tactile and visual influences are integrated with previously known wind-sensory mechanisms to achieve multisensory control of the full escape response.     

Gustatory projections from the nucleus of the solitary tract to the parabrachial nuclei in the hamster.

Taste-responsive cells in the nucleus of the solitary tract (NST) either project to the parabrachial nuclei (PbN) of the pons, through which taste information is transmitted to forebrain gustatory nuclei, or give rise to axons terminating locally within the medulla. Numerous anatomical studies clearly demonstrate a substantial projection from the rostral NST, where most taste-responsive cells are found, to the PbN. In contrast, previous electrophysiological studies in the rat have shown that only a small proportion (21-45%) of taste-responsive NST cells are antidromically activated from the PbN, suggesting that less than half the cells recorded from the NST are actually involved in forebrain processing of gustatory information. In the present experiment we investigated the projections from the NST to the PbN electrophysiologically in urethane anesthetized hamsters. Responses of 101 single neurons in the rostral NST were recorded extracellularly following lingual stimulation with 32 mM NaCl, sucrose and quinine hydrochloride (QHCl) and 3.2 mM citric acid. The taste-responsive region of the PbN was identified electrophysiologically and stimulated with a concentric bipolar electrode to antidromically activate each NST cell. Of the 101 taste-responsive NST cells, 81 (80.2%) were antidromically activated from the ipsilateral PbN. The mean firing rates to taste stimulation and the spontaneous activity of these projection neurons were significantly greater than those of non-projecting cells. Every sucrose-best neuron in the sample projected to the PbN. The mean conduction velocity of the 23 QHCl-best neurons was significantly lower than that of the other 58 PbN projection neurons, suggesting that the most QHCl-responsive cells are a subset of smaller neurons. These data show that a large majority of NST cells responsive to taste stimulation of the anterior tongue project to the gustatory subdivisions of the PbN and that these cells have the most robust responses to gustatory stimulation.