Airborne ascospores in Mérida (SW Spain) and the effect of rain and other meteorological parameters on their concentration

Airborne ascospores have been reported to be allergenic or plant pathogens, and their presence has traditionally been associated with rainfall events. The aim of the present study was to analyze the presence of airborne ascospores in relation to weather parameters in a town in SW Spain. A seven-day recording spore trap (Burkard) was used to sample the air over 2 years at 15 m above ground level on the terrace roof of the hospital in Mérida (SW Spain). Fungal spores were identified and counted by means of two longitudinal scans over the slides under ×1000 microscopy. A correlation analysis was made of the daily meteorological data and the airborne ascospore concentrations, and t-tests were used to compare data between the 2 years. Nineteen ascospore types were defined, including one-cell ascospores (Chaetomium, Diatrype, Helvella, Xylaria), tow-cell ascospores (Diaporthe, Mycosphaerella, Nectria, Venturia), transversally septate ascospores (Melanomma, Leptosphaeria, Paraphaeosphaeria, Sporormiella, Massaria), transversally and longitudinally septate ascospores (Pleospora), and ascospores within asci (Sordaria). Leptosphaeria consisted of a group of four types described according to the number of cells, hyaline grade, wall thickness, and ornamentation, and other ascospores comprised one last additional type. The average airborne ascospore concentration was 153 ascospores/m3. One-third each of this total were from the Leptosphaeria group, with an average 54 ascospores/m3, and the two-cell ascospores or Venturia-like group (Diaporthe, Mycosphaerella, Nectria, Venturia) with 51 ascospores/m3 on average. In third position was Pleospora with 27 ascospores/m3 on average. The month with highest concentration was September, with 238 ascospores/m3, and the lowest March, with 56 ascospores/m3. By seasons, autumn had the highest concentrations, followed by winter, spring, and summer. The maximum daily concentration reached was 3,371 ascospores/m3. Daily rainfall was significantly correlated with the ascospore types Diatrype, Mycosphaerella, Nectria, two subtypes of Leptosphaeria, and Pleospora. Relative humidity was positively correlated with those ascospore types and also with Diaporthe and Paraphaeosphaeria, and negatively with Chaetomium and Melanomma. The concentration was higher on rainy days than on days without rain for Pleospora, Leptosphaeria (3 subtypes), Diatrype, Diaporthe, Nectria, Mycosphaerella, and Paraphaeosphaeria. The daily temperatures were in general correlated with the same types as the relative humidity, but with the opposite sign. For the monthly data, there were no statistically significant differences between the 2 years studied.     

Airborne fungal spores and the thunderstorm of 24 June 1994

On the evening of 24 June 1994 there were thunderstorms which passed from the south to the north over large areas of England, followed by an extensive and initially alarming episode of 'thunderstorm asthma'. It was eventually concluded that the probable cause of this episode was the release of allergenic particles from wetted grass pollen. As part of the investigation of this episode changes in the fungal air spora were investigated at several sites. The fungal spores present varied from site to site. At London, Addlestone and Leicester there were very marked transient peaks of smut ustilospores (and to a much smaller extent at Cambridge), particularly those of Ustilago segetum (Bull.) Roussel. At many sites there were transient peaks of Cladosporium conidia. The possible causes of the transient peaks are discussed. At most of the other sites except London there were high concentrations of ascospores after the rain. Typically, Didymella ascospores occur after rain, but on this occasion they occurred at low concentrations at all sites, because of dry weather in the previous month. Instead, there were high concentrations of ascospores which normally occur in far lower numbers, viz. ascospores of Phaeosphaeria nigrans and Diatrypaceous ascospores. The reasons for this were related to increased rainfall in the previous thirteen months. There were site to site differences in the ascospore composition of the air spora, and this was related to habitat differences, where this was known. This revised version was published online in June 2006 with corrections to the Cover Date.     

The seasonal release of spores of Nectria galligena from apple cankers in Northern Ireland

Trapping of ascospores and conidia of Nectria galligena Bres. released from cankers on Bramley's Seedling apple trees over a period of three years showed that ascospores were most prevalent in the spring and early summer, and conidia from early summer to late autumn. Few ascospores were released in late summer or mid-winter but a minor period of discharge occurred in autumn. The distribution of cankers within trees in various orchards was also examined, and most were found to occur at the junctions of different seasons' growth. Possible reasons for this are discussed.     

Ecological studies of spores of aquatic hyphomycetes in the cringle brook,lincs

The aquatic Hyphomycete spora of the Cringle Brook, Lincs, was examined by foam sampling and by the use of cellophane impaction traps between August, 1968 and January, 1970.The species most frequently found (Tetracladium marchalianum, Alatospora acuminata and Flagellospora curvula) were generally in agreement with those found by other workers in temperate areas. Impaction trap samples generally contained fewer species than foam samples but filiform spore types such as Flagellospora were more frequently found on traps than in foam suggesting that impaction is more selective towards the filiform spore type than is foam. Many species increased in frequency in autumn accompanying and following leaf fall, and the winter spora was dominated by Alatospora acuminata, Clavariopsis aquatica, Clavatospora stellata, Flagellospora curvula and Lemonniera aquatica. During the summer the spora was dominated by Tetracladium marchalianum.The role of foam and impaction in the balance of aquatic spore populations is discussed in relation to techniques available for their study.     

Airborne fungi in an Italian rice mill

Summary Studies employing volumetric spore trap (VSP) and gravity settling culture plates (GSC) were conducted in order to analyse the air spora of a rice mill at Pavia, Italy, from October-December 1988. Results revealed a variety of fungal spores belonging to different genera and including recognized rice pathogenic fungi. The most frequent genera by GSC method includedAcremonium, Alternaria, Aspergillus, Aureobasidium, Cladosporium, Epicoccum, Fusarium, Helminthosporium, Mucor, Nigrospora, Penicillium, Rhizopus, Trichoderma, Trichothecium, and some unidentified fungi. Environmental assessment of fungal spores by VSP revealed that the most prevalent fungi were:Alternaria, Cladosporium, Epicoccum, Helminthosporium, Nigrospora, Pyricularia, Tilletia and hyaline, dark and coloured types of ascospores and basidiospores. Airborne fungal spore concentrations were particularly high (5,000–6,000 spores/m³) in the rooms of the rice mill where the initial stages of rough rice transformation take place, and dropped to 2,500 spores/m³ in the last room, where workers are. During a temporary interruption of the working processes, air spora concentration dropped below 1,000 spores/m³.Cladosporium, Epicoccum andNigrospora spores were predominant in all subdivisions of the indoor environments of the rice mill.     

Airborne Ascomycotina on the island of Crete: Seasonal patternsbased on an 8-year volumetric survey

An 8-year study was conducted on the island of Crete in order to identify airborne ascospores and to determine their seasonal pattern. A Burkard 7-day, volumetric spore-trap was continuously operated in the city of Irakleion – located in the center of the island – from 1994 through 2001. Relatively „high” ascospore counts (20 – 48 spores/m ³) were obtained from mid-spring through summer, while the rest of the year exhibited lower activity (8–16 spores/m³). The predominant ascospores identified were those of Leptosphaeria and Chaetomium; their concentrations varied from 1 or 2 spores up to a few dozens of spores/m³. Other spores encountered sporadically were: Ascobolus, Endophragmiella, Didymella, Diatrypaceae, Leptosphaerulina, Massaria, Pleospora, Sporormiella, Xylaria. The mean daily concentration of all identified ascospores was 30/m³ for the entire study period, corresponding to 13.9% of the total fungal load. Ascospores have been recognized as important inhalant allergens and have been implicated as contributing to symptoms of both rhinitis and asthma.     

Contribution of leaf surface fungi to the air spora

High concentrations of airborne fungal spores frequently occur from spring through fall in temperate areas of the world. Although it is generally assumed that fungi on leaf surfaces are contributors to the air spora, little data are available comparing the types of fungi found on leaf surfaces with those in the atmosphere. Air sampling was carried out with a Burkard Spore Trap located on the roof of a building on the University of Tulsa campus using standard methods. Leaf samples were aseptically collected from Ulmus americana and Quercus palustris trees on campus, placed in sterile plastic bags, and brought to the lab. For each leaf, 4 cm² areas of both upper and lower leaf surfaces were swabbed and plated on malt extract agar with streptomycin. Cultures were incubated at room temperature for 5–7 days and then examined microscopically. Results were expressed as colony forming units (CFU)/cm². Twenty-one fungal taxa were identified from the air samples. The most abundant taxa were Cladosporium, ascospores, basidiospores, and Alternaria; together these four spore types comprised over 90% of the yearly total. Yeasts were the most abundant fungi isolated from both leaf types. Among the mycelial fungi were Phoma species, followed by Cladosporium and Alternaria. Overall twenty genera of filamentous fungi were identified. Yeasts and Phoma are normally splash dispersed and were not identified in the Burkard air samples. However, 10 taxa isolated from leaf surfaces were registered in air samples. Crude estimates of the leaf surface area of each tree suggest that the total fungal load was approximately 5.04×10⁸ CFU for Ulmus and 2.71×10⁸ CFU for Quercus. Of these levels, 19% were from fungi also detected in air samples. The data suggest that some leaf-surface fungi are major contributors to the air spora.     

Selected airborne allergenic fungal spores and meteorological factors in Szczecin,Poland, 2004–2006

Airborne fungal spore concentrations in Szczecin, Poland, were studied between 2004 and 2006 with the objective of determining a seasonal variation in the concentrations of selected fungal spore types in relation to meteorological parameters. The presence of spores of five taxa, namely, Cladosporium, Ganoderma, Alternaria, Leptosphaeria and Didymella, was recorded using a volumetric method (Hirst type). Fungal spores were present in the air in large numbers during the summer, with the highest concentrations recorded mainly in June, July and August. The peak concentrations of two of the studied spore types, Ganoderma and Alternaria, occurred in August, while the concentrations of Cladosporium, Leptosphaeria and Didymella spores were the highest in July. Multiple regression analysis was performed for three fungal seasons—2004, 2005 and 2006. Spore concentration was found to be positively correlated with the minimum temperature. For some spore types, there was also a significant correlation between concentrations, relative humidity and rain.     

Correlation of spring spore concentrations and meteorological conditions in Tulsa, Oklahoma

Different spore types are abundant in the atmosphere depending on the weather conditions. Ascospores generally follow precipitation, while spore types such as Alternaria and Cladosporium are abundant in dry conditions. This project attempted to correlate fungal spore concentrations with meteorological data from Tulsa, Oklahoma during May 1998 and May 1999. Air samples were collected and analyzed by the 12-traverse method. The spore types included were Cladosporium, Alternaria, Epicoccum, Curvularia, Pithomyces, Drechslera, smut spores, ascospores, basidiospores, and other spores. Weather variables included precipitation levels, temperature, dew point, air pressure, wind speed, wind direction and wind gusts. There were over 242.57 mm of rainfall in May 1999 and only 64.01 mm in May 1998. The most abundant spore types during May 1998 and May 1999 were Cladosporium, ascospores, and basidiospores. Results showed that there were significant differences in the dry-air spora between May 1998 and May 1999. There were twice as many Cladosporium in May 1998 as in May 1999; both ascospores and basidiospores showed little change. Multiple regression analysis was used to determine which meteorological variables influenced spore concentrations. Results showed that there was no single model for all spore types. Different combinations of factors were predictors of concentration for the various fungi examined; however, temperature and dew point seemed to be the most important meteorological factors. Received: 5 July 2000 / Revised: 20 December 2000 / Accepted: 22 December 2000     

A national survey of airborne pollen and grass flowering in New Zealand,with implications for respiratory disorder

Airborne pollen and spore levels were monitored at seven sites in New Zealand using the Intermittent Cycling Rotorod sampler during the summer of 1988/1989. Grasses formed the major component of atmospheric pollen levels during spring and summer at every locality. Peak levels of grass and total pollen occurred during December or late November, with a slight latitudinal lag apparent at the more southern sites. Highest levels were recorded at the smaller rural centres of Gore and Kaikohe and the lowest at the larger urban centres of Auckland, Christchurch and Wellington. We make a first approximation of the likely risk to hayfever and allergic asthma patients at each of the seven centres. For example, significantly higher grass pollen levels were experienced at Kaikohe on 44% and 65% of days during November and December, compared with just 15% and 8% at Auckland. By recording the flowering seasons of the principal allergenic grass species at each locality, we determined the potentially allergenic grasses contributing to peak pollen levels, the most ubiquitous being tall fescue (Festuca arundinacea Schreb.), ryegrass (Lolium perenne L.,L. multiflorum Lam.), cocksfoot (Dactylis glomerata L.), Yorkshire fog (Holcus lanatus L.) and sweet vernal (Anthoxanthum odoratum L.). Corresponding author. Deceased.     

Phenological observations and trapping tactics for the granulate ambrosia beetle Xylosandrus crassiusculus (Coleoptera: Curculionidae,Scolytinae) in New Zealand

1. The granulate ambrosia beetle (GAB) Xylosandrus crassiusculus, was first formally detected in New Zealand in 2019. Since then, GAB has subsequently been found infesting numerous tree species in the Auckland region.
2. Flight intercept traps baited with ethanol lures were deployed from October 2019 to May 2021 at three sites in the Auckland region to ascertain the phenology of GAB in New Zealand. Two distinct peak flight periods were identified in early and late summer, while a smaller and inconsistent third peak was detected in early autumn. Logistical analysis of GAB captures in the ethanol-lured traps and degree-day (DD) accumulation indicated that 90% of flight activity is completed by 800 DD.
3. To assess monitoring tactics, flight intercept traps were baited with three different ethanol lures with varying release rates or ethanol-soaked or non-soaked wood bolts from three species of trees. A lure releasing 2 g ethanol per day was most effective at capturing GAB. Ethanol-soaked bolts were less effective than the lures.
4. Xylosandrus crassiusculus represents a significant risk for shrubs and trees native to New Zealand, as well as commercial horticultural and forestry trees. We recommend using ethanol-lured panel traps for monitoring purposes.

     

Seasonal changes in temperature response of photosynthesis and its contribution to annual carbon gain in Daphniphyllum humile,an evergreen understorey shrub

We evaluated seasonal variation in photosynthetic temperature dependence and its contribution to annual carbon gain in an evergreen understorey shrub, Daphniphyllum humile Maxim, growing at the forest border and in the understorey of a deciduous forest. Plants at both sites exhibited similar optimal temperatures for photosynthesis (T_opt). The activation energy for ribulose‐1,5‐bisphosphate (RuBP) carboxylation (H_aV) at both sites tended to be higher in summer than in spring or autumn, suggesting that H_aV may be the controlling factor in the T_opt shift in D. humile. In contrast to the seasonal changes in T_opt, the maximum photosynthetic rate at the optimal temperature (P_opt) differed between the two sites: it was lower in autumn than in summer at the forest border, but was the same in summer and autumn in the understorey. In the understorey plants, nitrogen content (N_area) increased in autumn, but this was not the case for forest border plants. In addition, Rubisco content increased significantly in autumn in the understorey leaves but decreased distinctly in forest border leaves. Increased N_area and Rubisco in understorey leaves resulted in increased in photosynthesis in autumn. Annual carbon gain was 30.8 mol·m^?2 in forest border leaves and 5.8 mol·m^?2 in understorey leaves. Carbon gain in understorey leaves during the short period after overstorey leaf fall and before snow accumulation was approximately 49% of annual carbon gain. Furthermore, autumn carbon gain calculated using activation energy of summer with autumn photosynthetic parameters underestimated the autumn carbon gain by as much as 31%. In conclusion, photosynthetic temperature acclimation may be a key factor in increasing annual carbon gain in understorey D. humile.     

A fungal spore calendar for the atmosphere of Melbourne, Australia, for the year 1993

A calendar of fungal spore seasons for Melbourne during 1993was established using a 7-day volumetric Burkard trap. Twenty-ninegenera and five spore groups were identified. The dominant spore typesdetected were Cladosporium (41.7%),Leptosphaeria (14.9%), Coprinus (14.6%),`Ascospore 1' (5.5%), Ganoderma (2.1%) andAlternaria (1.4%). Seasonally, spore levels ofCladosporium and Alternaria peaked in spring andsummer, Leptosphaeria and Ganoderma peaked towardsummer and autumn, `Ascospore 1' peaked in winter, whilst spore levelsof the basidiomycete Coprinus fluctuated year round. Inconclusion, a range of allergenic fungal spores were present in the airof Melbourne throughout the year.     

Overwintering of Sphaerotheca humuli, the cause of hop powdery mildew

The ability of Sphaerotheca humuli to overwinter as cleistocarps in infected hop cones and leaves and in aerial buds on rootstocks was examined during the winters of 1970-1, 1971-2 and 1972-3. Periodical examination of cleistocarps, collected in October and overwintered in Terylene bags on the soil of a hop garden, consistently revealed two periods of maturation ending in November and in March, when over 50% contained eight, well-defined ascospores. In laboratory tests cleistocarps, kept either in the hop garden or dry at 4, 8 or 18^oC during the winter, could not be encouraged to dehisce earlier than April when naturally dehisced cleistocarps were first detected in the field. More ascospores were discharged from cleistocarps, and germination of ascospores in laboratory tests was greater, at 18 than at 4, 8 or 24^oC. Colonies of S. humuli arose on leaves of potted plants exposed to overwintered cleistocarps in the hop garden and were observed microscopically to originate from ascospores. However, a Burkard spore trap, operated amidst the cleistocarps in this garden in 1972 and 1973, failed to detect ascospores. Ascospores, discharged onto susceptible leaves in the laboratory, germinated but failed to produce colonies. It was demonstrated that S. humuli can perennate in aerial, dormant buds on hop rootstocks. Examination of buds in autumn revealed mycelium external to and between the bud scales. At budburst the mycelium was still present internally. Cleistocarps were occasionally associated with hibernating mycelium. Primarily infected shoots arose from plants bearing infected buds in conditions which precluded chance infection. Some evidence was obtained that conditions during the winter determine the success of survival in buds. The fungus appeared to be incapable of infecting a selection of weeds common to hop gardens and their vicinity.     

Growth and yield of maize at different altitudes in Rhodesia

A late (SR 52) and an early (N × K) variety were grown, with irrigation, on well fertilized soil at three sites, Chiredzi (altitude 420 m), Henderson (1260 m) and Grasslands (1620 m). Mean incoming radiation was similar at all sites, while mean temperature decreased as altitude increased. Final total and grain dry weights were greatest at Henderson, and those of SR 52 exceeded those of N × K, although not greatly at Chiredzi. Initially, leaf area index (L) decreased with increase in altitude, but plants flowered later as altitude increased, and L at the time of flowering was greatest at Henderson. L at flowering was greater in SR 52 than in N × K, though only slightly so at Chiredzi. Leaves withered sooner after flowering at Chiredzi and Grasslands than at Henderson, but leaves of the two varieties withered at about the same time after flowering at each site. During most of the vegetative phase the efficiency of the leaves and crop growth rate (C) increased with decrease in altitude, and were greater in N × K than in SR 52. More dry matter was accumulated after flowering at Henderson than at the other sites, and more by SR 52 than by N × K at Henderson and Grasslands, but not at Chiredzi. Leaf area duration (D) after flowering was greater at Henderson than elsewhere, and greater in SR 52 than in N × K, except at Chiredzi. After flowering leaf efficiency (dry weight increase ÷D) was least at Henderson and greatest at Grasslands, but differed little between varieties at each site. The fraction in the grain of the dry matter accumulated after flowering decreased with increase in altitude and was greater in SR 52 than in N × K.     

北京河流底栖硅藻沿城乡梯度带空间分布及其季节变化

研究了北京市区沿城乡梯度带河流底栖硅藻的空间分布及其季节变化特征。通过在2014年的春季、夏季和秋季在山区对照溪流、城市上游河道和城市下游河道3个样区,共23个样点进行了底栖硅藻样品采集。分析表明,主要优势种的空间差异明显。在对照溪流,主要优势种为极小曲丝藻(Achnanthidium minutissimum);在城市上游河道,主要优势种分别为短文假十字脆杆藻(Pseudostaurosira brevistriata)和连结十字脆杆藻腹面变种(Staurosira construens var.venter);在城市下游河道,主要优势种为谷皮菱形藻(Nitzschia palea)。然而,3个样区内的主要优势种相对丰度季节变化较小(P0.05)。其中,A.minutissimum及P.brevistriata的季节变化特征均为春季秋季夏季,而S.construens var.venter及N.palea的季节变化特征均为春季夏季秋季。结果发现,北京市城乡梯度带的河流硅藻种类组成差异较大,但主要优势属种季节变化相对较小,表明硅藻种类能较好地反映城乡梯度带的河流环境变化状况,可用于北京市河流水质生物监测。     

Aeromycoflora of an agricultural farm in West Bengal,India: A five-year study (1994–1999)

A five-year (1994–1999) continuous survey of aeromycoflora had been carried out in an agricultural farm at a suburban area of Greater Calcutta. The sampling was carried out with Burkard volumetric spore trap placed 0.5?meter above the ground level at the centre in a small plot in a rice field. A total of 26 fungal spore types were identified microscopically. The most abundant types were Basidiospores (21.85–5.59%) followed by Cladosporium, Periconia, Nigrospora, Aspergilli group, Ascospores. The seasonal periodicity of the major dominant types had maxima in autumn, during the month of October. This coincided with the harvesting period of the rice crop during rainy season. In Petri plate exposure of nutrient media, different species of aspergilli group, Alternaria, Cladosporium, Curvularia, Nigrospora, and other taxa were identified. The diurnal periodicities recorded for different types were classified into night-time, post dawn, middle day and double maxima (showing two peaks in a day) patterns. In general, no significant correlation between the total air spora and the meteorological parameters could be established. It is evident that the local cropping practices may have some regulating role on spore concentration in the air of the study area.     

Some components of the air spora in Jamaica and their possible medical application

A knowledge of the pollen and fungal spores which comprise the air spora is useful as a preliminary approach to the problem of respiratory allergy. Therefore, this study of the qualitative and quantitative aspects of the air spora was done. Fungal spores were found to be numerically dominant, comprising 97.73% whilst pollen comprised 0.40% of the total material observed. A small number of types made up the majority of the fungal air spora, namely, Cladosporium, the Sporobolomycetaceae group, Diatrype, Glomerella, hyaline and coloured basidiospores, and septate fusiform spores. Seasonal periodicity studies on twenty-five fungal types showed that a high number of spores were trapped for sixteen during wet months, four during cooler months, and that five showed no seasonal trends. Mean diurnal periodicity studies for the year on the same twenty-five spore types showed that all had a maximum number of spores trapped at some time during the day. Investigation of the effect of rainfall on the numbers of spores released showed that the amount and duration of rainfall, the time of day rain occurs, and the length of the dry period preceding rain were of varying importance to particular spore types.     

On factors influencing fruit-set and sterility in arecanut (Areca Catechu Linn.)

Summary (1) Histological studies on the stigmas of female flowers of areca indicate the possibility of the stigmatic surfaces being impacted upon by air spora. The presence of spores or hyphae together with pollen grains has been shown in stigmatic surface.(2) Air spora of arecanut plantation chiefly consist ofAspergillus, Penicillium, Actinomycetes, Mucor, Alternaria, Cladosporium andBacteria.(3) The types of fungi, bacteria and actinomycetes adhering to the stigma are isolated.(4) The metabolites of the fungi, bacteria and actinomycetes isolated from the stigmas of arecanut flowers inhibit,in vitro, considerably the germination of pollen grains and rate of growth of pollen tubes. In certain instances there is no germination of the pollen grains in the metabolites of fungi, bacteria and actinomycetes. In view of the results the significance of the air spora in affecting the normal course of pollination and fertilization is discussed.     

Airborne ascospores in Tetouan (NW Morocco) and meteorological parameters

Ascospores are frequently found as airborne fungal spores and recognized in various areas as an important cause of respiratory allergies. The main objective of the study was to determine the relationship between airborne ascospores and meteorological parameters using multivariate canonical correspondence analysis (CCA) and Spearman correlation. The aerobiological monitoring of fungal spores was performed over 5 years (2009–2013) using a Burkard volumetric spore traps. Seven main types of ascospores were identified: Leptosphaeria, Pleospora, Venturia, Diatrype, Chaetomium, Sporormiella and Ascobolus. The CCA results showed that all applied variables accounted for 27.4 % of the total variance in the spore data in the 5 years. The largest contribution to the total variance was explained in this period by the maximum air temperature (10.3 %). The effect of meteorological factors varied among years. The highest values of the total variance in the spore data, explained by the statistically significant variables, were observed in 2012 (28.6 %), with the highest contribution to minimum relative humidity (8.0 %). Most ascospores showed positive and statistically significant correlation with relative humidity and rainfall. In contrast, ascospores of Chaetomium were negatively correlated with precipitation and the relative humidity and positively with temperature. Based on these results, epidemiological and allergological studies must deserve more attention to estimate the allergenic potential of the ascospores.