Seasonal variations of triterpene acid contents in Viscum album L. on typical host trees of Hyrcanian forests.

Viscum album L. (mistletoe) is a semiparasitic plant of the Santalaceae family. A valuable group of bioactive compounds in mistletoe are triterpene acids (TTAs), which possess anti-inflammatory and anticancer properties. Parrotia persica and Carpinus betulus are the most common hosts of mistletoe in the Hyrcanian forests of Iran. This study was performed to compare the content of oleanolic acid (OA), betulinic acid (BA), and ursolic acid (UA) in the mistletoe foliage (stems and leaves) from P. persica and C. betulus in various seasons for the first time. The results showed that OA was the prevailing TTA in all samples, while UA was found in none of them. The maximum amount of OA (12.38 mg/g dry weight [DW]) and BA (1.68 mg/g DW) was detected in V. album from P. persica in summer. The minimum amount of OA (5.58 mg/g DW) and BA (0.72 mg/g DW) was observed in that growing on C. betulus in winter. However, the mistletoe from C. betulus showed the greatest level of OA in spring (9.06 mg/g DW) and BA in summer and autumn (0.92 and 0.97 mg/g DW, respectively). The data collected in this study complement existing research on this subject from around the world.

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions.

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

Beech tree masting explains the inter-annual variation in the fall and spring peaks of Ixodes ricinus ticks with different time lags.

BACKGROUND: The tick Ixodes ricinus is an important vector of tick-borne diseases including Lyme borreliosis. In continental Europe, the nymphal stage of I. ricinus often has a bimodal phenology with a large spring peak and a smaller fall peak. There is consensus about the origin of the spring nymphal peak, but there are two alternative hypotheses for the fall nymphal peak. In the direct development hypothesis, larvae quest as nymphs in the fall of the same year that they obtained their larval blood meal. In the developmental diapause hypothesis, larvae overwinter in the engorged state and quest as nymphs one year after they obtained their larval blood meal. These two hypotheses make different predictions about the time lags that separate the larval blood meal and the density of questing nymphs (DON) in the spring and fall. METHODS: Inter-annual variation in seed production (masting) by deciduous trees is a time-lagged index for the density of vertebrate hosts (e.g., rodents) which provide blood meals for larval ticks. We used a long-term data set on the masting of the European beech tree and a 15-year study on the DON at 4 different elevation sites in western Switzerland to differentiate between the two alternative hypotheses for the origin of the fall nymphal peak. RESULTS: Questing I. ricinus nymphs had a bimodal phenology at the three lower elevation sites, but a unimodal phenology at the top elevation site. At the lower elevation sites, the DON in the fall was strongly correlated with the DON in the spring of the following year. The inter-annual variation in the densities of I. ricinus nymphs in the fall and spring was best explained by a 1-year versus a 2-year time lag with the beech tree masting index. Fall nymphs had higher fat content than spring nymphs indicating that they were younger. All these observations are consistent with the direct development hypothesis for the fall peak of I. ricinus nymphs at our study site. Our study provides new insight into the complex bimodal phenology of this important disease vector. CONCLUSIONS: Public health officials in Europe should be aware that following a strong mast year, the DON will increase 1 year later in the fall and 2 years later in the spring. Studies of I. ricinus populations with a bimodal phenology should consider that the spring and fall peak in the same calendar year represent different generations of ticks.

Australian mosquito assemblages vary between ground and sub-canopy habitats.

BACKGROUND: The surveillance and control of mosquito-borne diseases is dependent upon understanding the bionomics and distribution of the vectors. Most studies of mosquito assemblages describe species abundance, richness and composition close to the ground defined often by only one sampling method. In this study, we assessed Australian mosquito species near the ground and in the sub-canopy using two traps baited with a variety of lures. METHODS: Mosquitoes were sampled using a 4 × 4 Latin square design at the Cattana Wetlands, Australia from February to April 2020, using passive box traps with octenol and carbon dioxide and three variations of a sticky net trap (unbaited, and baited with octenol or octenol and carbon dioxide). The traps were deployed at two different heights: ground level (≤ 1 m above the ground) and sub-canopy level (6 m above the ground). RESULTS: In total, 27 mosquito species were identified across the ground and sub-canopy levels from the different traps. The abundance of mosquitoes at the ground level was twofold greater than at the sub-canopy level. While the species richness at ground and sub-canopy levels was not significantly different, species abundance varied by the collection height. CONCLUSIONS: The composition of mosquito population assemblages was correlated with the trap types and heights at which they were deployed. Coquillettidia species, which prefer feeding on birds, were mainly found in the sub-canopy whereas Anopheles farauti, Aedes vigilax and Mansonia uniformis, which have a preference for feeding on large mammals, were predominantly found near the ground. In addition to trap height, environmental factors and mosquito bionomic characteristics (e.g. larval habitat, resting behaviour and host blood preferences) may explain the vertical distribution of mosquitoes. This information is useful to better understand how vectors may acquire and transmit pathogens to hosts living at different heights.

Pest detection dogs for wood boring longhorn beetles.

Invasive alien species are increasing due to globalization. Their spread has resulted in global economic losses. Asian [Anoplophora glabripennis (Motschulsky)] (ALB) and citrus [A. chinensis (Forster)] (CLB) longhorn beetles are two introduced wood borers which contribute to these economic losses e.g. the destruction of tree plantations. Early detection is key to reduce the ecological influence alongside the detrimental and expensive eradication. Dogs (Canis lupus familiaris) can detect these insects, potentially at an early stage. We trained two privately owned dogs to investigate their use as detection tools. We tested the dog's ability to discriminate ALB and CLB from native wood borers by carrying out double-blind and randomized experiments in three search conditions; (1) laboratory, (2) semi-field and (3) standardized field. For condition one, a mean sensitivity of 80%, specificity of 95% and accuracy of 92% were achieved. For condition two and three, a mean sensitivity of 88% and 95%, specificity of 94% and 92% and accuracy of 92% and 93% were achieved. We conclude that dogs can detect all types of traces and remains of ALB and CLB and discriminate them from native wood borers and uninfested wood, but further tests on live insects should be initiated.

The relationship between resource abundance and insect herbivory on islands.

We examined the relationship between resource abundance and the feeding activity of phytophagous insects on three common island plants. The aim was to investigate the correlation between phytophagous insects' abundance and availability of food and island geography. We collected 30,835 leaves from three tree species groups (Mallotus japonicus, Prunus species, and Quercus species) on 18 islands in southwest Korea. The number of plant resources for herbivores varied: the number of leaves per shoot was the highest in Mallotus, leaf weight and the water content per leaf was significantly lower in Quercus species. External feeding was higher for Prunus and Quercus species, whereas the internal feeding type was significantly higher for Quercus species. Geography (area and distance), elevation and food resource (elevation, number of plant species, and the forest cover rate) had a variable effect on phytophagous insects feeding activities: distance and the number of plant species were more explainable to the external feeding guild. In contrast, area and forest cover were more to the internal feeding guild.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Synthesis and Insecticidal Evaluation of Chiral Neonicotinoids Analogs: The Laurel Wilt Case.

Xyleborus sp beetles are types of ambrosia beetles invasive to the United States and recently also to Mexico. The beetle can carry a fungus responsible for the Laurel Wilt, a vascular lethal disease that can host over 300 tree species, including redbay and avocado. This problem has a great economic and environmental impact. Indeed, synthetic chemists have recently attempted to develop new neonicotinoids. This is also due to severe drug resistance to "classic" insecticides. In this research, a series of neonicotinoids analogs were synthesized, characterized, and evaluated against Xyleborus sp. Most of the target compounds showed good to excellent insecticidal activity. Generally, the cyclic compounds also showed better activity in comparison with open-chain compounds. Compounds R-13, 23, S-29, and 43 showed a mortality percent of up to 73% after 12 h of exposure. These results highlight the enantioenriched compounds with absolute R configuration. The docking results correlated with experimental data which showed both cation-π interactions in relation to the aromatic ring and hydrogen bonds between the search cavity 3C79 and the novel molecules. The results suggest that these sorts of interactions are responsible for high insecticidal activity.

To the canopy and beyond: Air dispersal as a mechanism of ubiquitous protistan pathogen assembly in tree canopies.

Cercozoa and Oomycota contain a huge biodiversity and important pathogens of forest trees and other vegetation. We analyzed air dispersal of these protistan phyla with an air sampler near-ground (~2 m) and in tree crowns (~25 m) of three tree species (oak, linden and ash) in a temperate floodplain forest in March (before leafing) and May (after leaf unfolding) 2019 with a cultivation-independent high-throughput metabarcoding approach. We found a high diversity of Cercozoa and Oomycota in air samples with 122 and 81 OTUs, respectively. Especially oomycetes showed a significant difference in community composition between both sampling dates. Differences in community composition between air samples in tree canopies and close to the ground were however negligible, and also tree species identity did not affect communities in air samples, indicating that the distribution of protistan propagules through the air was not spatially restricted in the forest ecosystem. OTUs of plant pathogens, whose host species did not occur in the forest, demonstrate dispersal of propagules from outside the forest biome. Overall, our results lead to a better understanding of the stochastic processes of air dispersal of protists and protistan pathogens, a prerequisite to understand the mechanisms of their community assembly in forest ecosystems.

Distribution pattern of entry holes of the tree-killing bark beetle Polygraphus proximus.

Bark beetles attack their hosts at uniform intervals to avoid intraspecific competition in the phloem. Bark texture and phloem thickness also affect bark beetle attacks, and the bark characteristics are not spatially homogeneous; therefore, the distribution patterns of entry holes can demonstrate an aggregated distribution. Polygraphus proximus Blandford (Coleoptera: Scolytinae) is a non-aggressive phloephagous bark beetle that feeds on Far Eastern firs. They have caused mass mortality in Russia and Japan. However, the distribution pattern of entry holes of P. proximus and spatial relationships with bark characteristics have not been studied. Thus, we investigated the distribution pattern of entry holes of P. proximus. The distribution of entry holes was significantly uniform in most cases. As the attack density increased, an aggregated distribution pattern within a short distance (< 4.0 cm) was observed. The rough bark had a significantly higher number of entry holes than the remaining bark. The distribution pattern of entry holes demonstrated a significantly aggregated spatial association with rough bark. Finally, rough bark around knots had significantly thicker phloem than the remaining barks. These suggest that P. proximus may preferentially attack rough bark to reproduce in the thicker phloem under a rough bark surface.

Biotic and abiotic factors causing the collapse of Robinia pseudoacacia L. veteran trees in urban environments.

Robinia pseudoacacia L. is an interesting example of how one plant species can be considered invasive or useful depending on its environment. In the past this tree species was planted for decorative purposes and for wood in Poland. For many years it was recommended in poor and degraded habitats because it facilitated late-successional plant species. The aim of this study was to verify if black locust can still be regarded as a resistant tree species in urban greenery. The health condition of old tree specimens growing along streets and in parks was compared. The occurrence of pests and pathogens on R. pseudoacacia trees was assessed and the content of mineral elements in leaves was examined. The research results showed that the health of black locust trees growing in the urban environment in Polish cities, especially along streets (in comparison to park sites), deteriorated significantly due to the interaction of harmful biotic and abiotic factors. Increased level of toxic metals (Fe, Zn, Pb, Mn and Cd) in plant tissues and the accumulation of pests and pathogens negatively influenced the health of R. pseudoacacia.

Cross-scale interaction of host tree size and climatic water deficit governs bark beetle-induced tree mortality.

The recent Californian hot drought (2012-2016) precipitated unprecedented ponderosa pine (Pinus ponderosa) mortality, largely attributable to the western pine beetle (Dendroctonus brevicomis; WPB). Broad-scale climate conditions can directly shape tree mortality patterns, but mortality rates respond non-linearly to climate when local-scale forest characteristics influence the behavior of tree-killing bark beetles (e.g., WPB). To test for these cross-scale interactions, we conduct aerial drone surveys at 32 sites along a gradient of climatic water deficit (CWD) spanning 350 km of latitude and 1000 m of elevation in WPB-impacted Sierra Nevada forests. We map, measure, and classify over 450,000 trees within 9 km2, validating measurements with coincident field plots. We find greater size, proportion, and density of ponderosa pine (the WPB host) increase host mortality rates, as does greater CWD. Critically, we find a CWD/host size interaction such that larger trees amplify host mortality rates in hot/dry sites. Management strategies for climate change adaptation should consider how bark beetle disturbances can depend on cross-scale interactions, which challenge our ability to predict and understand patterns of tree mortality.

Phytophthora Species Associated with Roots of Native and Non-native Trees in Natural and Managed Forests.

Roots act as a biological filter that exclusively allows only a portion of the soil-associated microbial diversity to infect the plant. This microbial diversity includes organisms both beneficial and detrimental to plants. Phytophthora species are among the most important groups of detrimental microbes that cause various soil-borne plant diseases. We used a metabarcoding approach with Phytophthora-specific primers to compare the diversity and richness of Phytophthora species associated with roots of native and non-native trees, using different types of soil inocula collected from native and managed forests. Specifically, we analysed (1) roots of two non-native tree species (Eucalyptus grandis and Acacia mearnsii) and native trees, (2) roots of two non-native tree species from an in vivo plant baiting trial, (3) roots collected from the field versus those from the baiting trial, and (4) roots and soil samples collected from the field. The origin of the soil and the interaction between root and soil significantly influenced Phytophthora species richness. Moreover, species richness and community composition were significantly different between the field root samples and field soil samples with a higher number of Phytophthora species in the soil than in the roots. The results also revealed a substantial and previously undetected diversity of Phytophthora species from South Africa.

Tree canopy arthropods have idiosyncratic responses to plant ecophysiological traits in a warm temperate forest complex.

Biodiversity studies on forest canopies often have narrow arthropod taxonomic focus, or refer to a single species of tree. In response, and to better understand the wide range of drivers of arthropod diversity in tree canopies, we conducted a large-scale, multi-taxon study which (a) included effect of immediate surroundings of an individual tree on plant physiological features, and (b), how these features affect compositional and functional arthropod diversity, in a warm, southern Afro-temperate forest. We found that tree species differed significantly in plant physiological features and arthropod diversity patterns. Surprisingly, we found negative correlation between surrounding canopy cover, and both foliar carbon and arthropod diversity in host trees, regardless of tree species. Subtle, tree intraspecific variation in physiological features correlated significantly with arthropod diversity measures, but direction and strength of correlations differed among tree species. These findings illustrate great complexity in how canopy arthropods respond to specific tree species, to immediate surroundings of host trees, and to tree physiological features. We conclude that in natural forests, loss of even one tree species, as well as homogenization of the crown layer and/or human-induced environmental change, could lead to profound and unpredictable canopy arthropod biodiversity responses, threatening forest integrity.

Did Mammals Bring the First Mistletoes into the Treetops?

AbstractThe growth habit of mistletoes, the only woody, parasitic plants to infect host canopies, represents a key innovation. How this aerially parasitic habit originated is unknown; mistletoe macrofossils are relatively recent, from long after they adapted to canopy life and evolved showy, bird-pollinated flowers; sticky, bird-dispersed seeds; and woody haustoria diverting water and nutrients from host branches. Since the transition to aerial parasitism predates the origin of mistletoes' contemporary avian seed dispersers by 20-40 million years, this leaves unanswered the question of who the original mistletoe dispersers were. By integrating fully resolved phylogenies of mistletoes and aligning the timing of historic events, I identify two ancient mammals as likely candidates for planting Viscaceae and Loranthaceae in the canopy. Just as modern mouse lemurs and galagos disperse viscaceous mistletoe externally (grooming the sticky seeds from their fur), Cretaceous primates (e.g., Purgatorius) may have transported seeds of root-parasitic understory shrubs up into the canopy of Laurasian forests. In the Eocene, ancestors of today's mistletoe-dispersing marsupials, Dromiciops, likely fed on the nutritious fruit of root-parasitic loranthaceous shrubs, depositing the seeds atop western Gondwanan forest crowns. Once mistletoes colonized the canopy, subsequent evolution and diversification coincided with the rise of nectar- and fruit-dependent birds.

Unravelling the gut bacteriome of Ips (Coleoptera: Curculionidae: Scolytinae): identifying core bacterial assemblage and their ecological relevance.

Bark beetles often serve as forest damaging agents, causing landscape-level mortality. Understanding the biology and ecology of beetles are important for both, gathering knowledge about important forest insects and forest protection. Knowledge about the bark beetle gut-associated bacteria is one of the crucial yet surprisingly neglected areas of research with European tree-killing bark beetles. Hence, in this study, we survey the gut bacteriome from five Ips and one non-Ips bark beetles from Scolytinae. Results reveal 69 core bacterial genera among five Ips beetles that may perform conserved functions within the bark beetle holobiont. The most abundant bacterial genera from different bark beetle gut include Erwinia, Sodalis, Serratia, Tyzzerella, Raoultella, Rahnella, Wolbachia, Spiroplasma, Vibrio, and Pseudoxanthomonas. Notable differences in gut-associated bacterial community richness and diversity among the beetle species are observed. Furthermore, the impact of sampling location on the overall bark beetle gut bacterial community assemblage is also documented, which warrants further investigations. Nevertheless, our data expanded the current knowledge about core gut bacterial communities in Ips bark beetles and their putative function such as cellulose degradation, nitrogen fixation, detoxification of defensive plant compounds, and inhibition of pathogens, which could serve as a basis for further metatranscriptomics and metaproteomics investigations.

An early gall-inducing parasitic wasp adversely affects the fitness of its host Ficus tree but not the pollinator.

The fig tree-fig pollinator mutualism is one of the most tightly knit symbiotic systems. The research on the ecology of non-pollinators which exploit the mutualism without providing services to the host is very limited and conclusions about the role they play in the maintenance of this mutualism are full of contradictions. The non-pollinating fig wasps species are highly diverse in their feeding habit and ecological function, which may result in different consequences on the mutualism. Sycophaga testacea is an early-ovipositing galler hosted by Ficus racemosa, which is a potencial competitor to the pollinators as they use the same female flowers in the fig as their ovipositing sites. In this study, we investigate the effect of S. testacea on the production of both pollinator and fig tree with a field control experiment. Seed production is decreased significantly when the figs were parasitized, while the offspring production of the pollinator is not significantly affected, which indicates that this galler species has a harmful effect on the fitness of its host fig tree but not the pollinator. The overall development ratio of the galls is decreased significantly when the figs were parasitized, and we found that the intrinsic low development ratio of S. testacea is responsible for the decrease in the overall development ratio.

Seasonal changes in population structure of the ambrosia beetle Xylosandrus compactus and its associated fungi in a southern Mediterranean environment.

Exotic ambrosia beetles are increasing in Europe due to global trade and global warming. Among these xylomycetophagous insects, Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae) is a serious threat for several Mediterranean host plants. Carob trees growing in Sicily (Italy) have been extensively attacked by beetles leading to rapid tree decline. Although X. compactus has been found in Europe for several years, most aspects of its ecology are still unknown. We thus studied the population structure and dynamics of X. compactus, together with its twig size preference during a sampling of infested carob trees in south east Sicily. In addition, fungi associated with insects or galleries were isolated and characterized. The results showed that, in this newly-colonized environment and host plant, adult X. compactus overwinters inside twigs and starts to fly and reproduce in mid spring, completing five generations before overwintering in late fall. The mean diameter of carob twigs infested by the beetle varied significantly over the seasons, with the insect tending to infest larger twigs as season progresses. The mean number of adults/gallery was 19.21, ranging from 6 to 28. The minimum temperature significantly affected the overwintering adult mortality. Ambrosiella xylebori and Fusarium solani were the main symbionts associated with the pest in this study. Acremonium sp. was instead recorded for the first time in Europe inside X. compactus galleries. Several other fungi species were also found for the first time in association with X. compactus. Our findings provide useful insights into the sustainable management of this noxious pest.

Ground-dwelling arthropods of pinyon-juniper woodlands: Arthropod community patterns are driven by climate and overall plant productivity, not host tree species.

Pinyon-juniper (PJ) woodlands have drastically changed over the last century with juniper encroaching into adjacent habitats and pinyon experiencing large-scale mortality events from drought. Changes in climate and forest composition may pose challenges for animal communities found in PJ woodlands, especially if animals specialize on tree species sensitive to drought. Here we test habitat specialization of ground-dwelling arthropod (GDA) communities underneath pinyon and juniper trees. We also investigate the role of climate and productivity gradients in structuring GDAs within PJ woodlands using two elevational gradients. We sampled 12,365 individuals comprising 115 taxa over two years. We found no evidence that GDAs differ under pinyon or juniper trees, save for a single species of beetle which preferred junipers. Climate and productivity, however, were strongly associated with GDA communities and appeared to drive differences between sites. Precipitation was strongly associated with arthropod richness, while differences in GDA composition were associated with environmental variables (precipitation, temperature, vapor pressure, and normalized difference vegetation index). These relationships varied among different arthropod taxa (e.g. ants and beetles) and community metrics (e.g. richness, abundance, and composition), with individual taxa also responding differently. Overall, our results suggest that GDAs are not dependent on tree type, but are strongly linked to primary productivity and climate, especially precipitation in PJ woodlands. This implies GDAs in PJ woodlands are more susceptible to changes in climate, especially at lower elevations where it is hot and dry, than changes in dominant vegetation. We discuss management implications and compare our findings to GDA relationships with vegetation in other systems.

Modelling the dynamics of Pine Wilt Disease with asymptomatic carriers and optimal control.

Pine wilt disease is a lethal tree disease caused by nematodes carried by pine sawyer beetles. Once affected, the trees are destroyed within a few months, resulting in significant environmental and economic losses. The role of asymptomatic carrier trees in the disease dynamics remains unclear. We developed a mathematical model to investigate the effect of asymptomatic carriers on the long-term outcome of the disease. We performed a stability and sensitivity analysis to identify key parameters and used optimal control to examine several intervention options. Our model shows that, with the application of suitable controls, the disease can be eliminated in the vector population and all tree populations except for asymptomatic carriers. Of the possible controls (tree injection, elimination of infected trees, insecticide spraying), we determined that elimination of infected trees is crucial. However, if the costs of insecticide spraying increase, it can be supplemented (although not replaced entirely) by tree injection, so long as some spraying is still undertaken.