Similarity Of Parasite Communities Research Articles

Host ecology and biogeography drive parasite community composition in Atlantic killifishes.

Understanding the mechanisms of parasite community assembly can be confounded by phylogenetic distance among host species. Addressing this requires focusing on parasite communities within closely related taxa. Thus, we took a macroecological approach to examining parasite community structure within Killifish species in the genus Fundulus to disentangle the effects of host phylogeny and ecological variables. We constructed a database of parasite communities within Fundulus species from 15 published and unpublished surveys covering the Atlantic coast of the US and Canada. The database was expanded by sampling sites in underrepresented provinces and states, totaling 10 Fundulus species from 57 unique geographic sites. Univariate analysis of observed parasite species richness among Fundulus populations in the dataset found that latitude, climate type, and salinity were the dominant factors determining parasite species richness. Multivariate analysis found that host species and landscape type were the most important factors in determining the similarity of parasite assemblages. Unexpectedly, parasite species richness decreased in low latitudes, and host phylogenetic distance was not found to be a significant factor in the similarity of parasite communities. These results indicate that commonly reported large-scale drivers of parasite community structure, such as latitude and phylogeny, could have diminished significance at the host genus level relative to host ecology, biogeography, and local landscape factors.

Within-host and external environments differentially shape β-diversity across parasite life stages.

Uncovering drivers of community assembly is a key aspect of learning how biological communities function. Drivers of community similarity can be especially useful in this task as they affect assemblage-level changes that lead to differences in species diversity between habitats. Concepts of β-diversity originally developed for use in free-living communities have been widely applied to parasite communities to gain insight into how infection risk changes with local conditions by comparing parasite communities across abiotic and biotic gradients. Factors shaping β-diversity in communities of immature parasites, such as larvae, are largely unknown. This is a key knowledge gap as larvae are frequently the infective life-stage and understanding variation in these larval communities is thus key for disease prevention. Our goal was to uncover links between β-diversity of parasite communities at different life stages; therefore, we used gastrointestinal nematodes infecting African buffalo in Kruger National Park, South Africa, to investigate within-host and extra-host drivers of adult and larval parasite community similarity. We employed a cross-sectional approach using PERMANOVA that examined each worm community at a single time point to assess independent drivers of β-diversity in larvae and adults as well as a longitudinal approach with path analysis where adult and larval communities from the same host were compared to better link drivers of β-diversity between these two life stages. Using the cross-sectional approach, we generally found that intrinsic, within-host traits had significant effects on β-diversity of adult nematode communities, while extrinsic, extra-host variables had significant effects on β-diversity of larval nematode communities. However, the longitudinal approach provided evidence that intrinsic, within-host factors affected the larval community indirectly via the adult community. Our results provide key data for the comparison of community-level processes where adult and immature stages inhabit vastly different habitats (i.e. within-host vs. abiotic environment). In the context of parasitism, this helps elucidate host infection risk via larval stages and the drivers that shape persistence of adult parasite assemblages, both of which are useful for predicting and preventing infectious disease.

Decay of parasite community similarity with host phylogenetic and geographic distances among deep-sea fish (grenadiers).

Although parasite community studies are growing in numbers, our understanding of which macro-ecological and evolutionary processes have shaped parasite communities is still based on a narrow range of host–parasite systems. The present study assessed the diversity and endoparasite species composition in New Zealand deep-sea fish (grenadiers, family Macrouridae), and tested the effects of host phylogeny and geography on the structure of endoparasite communities using a distance decay framework. We found that grenadiers from the Chatham Rise harboured a surprisingly high diversity of digeneans, cestodes and nematodes, with different species of grenadiers having different parasite assemblages. Our results demonstrate that community similarity based on the presence/absence of parasites was only affected by the phylogenetic relatedness among grenadier species. In contrast, both phylogenetic distance among grenadiers (measured as the number of base-pair differences of DNA sequences) and geographic distance between sample locations influenced the similarity of parasite communities based on the parasites' prevalence and mean abundance. Our key findings highlight the significant effect of deep-sea host phylogeny in shaping their parasite assemblages, a factor previously neglected in studies of parasite communities in deep-sea systems.

Non-native gobies share predominantly immature parasites with local fish hosts

Non-native species are known to escape their parasites following introduction into a new range, but they also often acquire local parasites as a function of time since establishment. We compared the parasite faunas of five non-native Ponto-Caspian gobies (Gobiidae) and local fish species (Perca fluviatilis, Gymnocephalus cernua, Gobio gobio) in three European river systems; the Rivers Rhine, Vistula and Morava, where Ponto-Caspian gobies were introduced 4-13 years prior to the study. Overall parasite species richness was considerably lower in non-native gobies compared to local fish species, and the same result was found at the component and infra-community levels. Both parasite abundance and diversity greatly varied among the regions, with the highest values found in the River Vistula (Wloclawski Reservoir), compared to a relatively impoverished parasite fauna in the River Morava (Danube basin). While only half of parasite species found in local hosts were acquired by non-native gobies, most of the parasites found in gobies were shared with local fish species related either phylogenetically (percids) or ecologically (benthic gudgeon), including the co-introduced monogenean Gyrodactylus proterorhini. As a result, similarity in parasite communities strongly reflected regional affiliation, while phylogenetic distances between fish host species did not play a significant role in parasite community composition. In accordance with other studies, all parasites acquired by gobies in their new range were generalists, all of them infecting fish at the larval/subadult stage, indicating the possible importance of gobies in the life cycle of euryxenous parasites. The absence of adult generalists, particularly ectoparasites with low host specificity, in non-native fish may reflect their generally low abundance in the environment, while an absence of adult endoparasitic generalists was probably related to other factors.

Parasite sharing in wild ungulates and their predators: Effects of phylogeny, range overlap, and trophic links.

Understanding factors that facilitate interspecific pathogen transmission is a central issue for conservation, agriculture, and human health. Past work showed that host phylogenetic relatedness and geographical proximity can increase cross-species transmission, but further work is needed to examine the importance of host traits, and species interactions such as predation, in determining the degree to which parasites are shared between hosts. Here we consider the factors that predict patterns of parasite sharing across a diverse assemblage of 116 wild ungulates (i.e., hoofed mammals in the Artiodactyla and Perissodactyla) and nearly 900 species of micro- and macroparasites, controlling for differences in total parasite richness and host sampling effort. We also consider the effects of trophic links on parasite sharing between ungulates and carnivores. We tested for the relative influence of range overlap, phylogenetic distance, body mass, and ecological dissimilarity (i.e., the distance separating species in a Euclidean distance matrix based on standardized traits) on parasite sharing. We also tested for the effects of variation in study effort as a potential source of bias in our data, and tested whether carnivores reported to feed on ungulates have more ungulate parasites than those that use other resources. As in other groups, geographical range overlap and phylogenetic similarity predicted greater parasite community similarity in ungulates. Ecological dissimilarity showed a weak negative relationship with parasite sharing. Counter to our expectations, differences, not similarity, in host body mass predicted greater parasite sharing between pairs of ungulate hosts. Pairs of well-studied host species showed higher overlap than poorly studied species, although including sampling effort did not reduce the importance of biological traits in our models. Finally, carnivores that feed on ungulates harboured a greater richness of ungulate helminths. Overall, we show that the factors that predict parasite sharing in wild ungulates are similar to those known for other mammal groups, and demonstrate the importance of controlling for heterogeneity in host sampling effort in future analyses of parasite sharing. We also show that ecological interactions, in this case trophic links via predation, can allow sharing of some parasite species among distantly related host species.

Host traits associated with species roles in parasite sharing networks

The community of host species that a parasite infects is often explained by functional traits and phylogeny, predicting that closely related hosts or those with particular traits share more parasites with other hosts. Previous research has examined parasite community similarity by regressing pairwise parasite community dissimilarity between two host species against host phylogenetic distance. However, pairwise approaches cannot target specific host species responsible for disproportionate levels of parasite sharing. To better identify why some host species contribute differentially to parasite diversity patterns, we represent parasite sharing using ecological networks consisting of host species connected by instances of shared parasitism. These networks can help identify host species and traits associated with high levels of parasite sharing that may subsequently identify important hosts for parasite maintenance and transmission within communities. We used global‐scale parasite sharing networks of ungulates, carnivores, and primates to determine if host importance – encapsulated by the network measures degree, closeness, betweenness, and eigenvector centrality – was predictable based on host traits. Our findings suggest that host centrality in parasite sharing networks is a function of host population density and range size, with range size reflecting both species geographic range and the home range of those species. In the full network, host taxonomic family became an important predictor of centrality, suggesting a role for evolutionary relationships between host and parasite species. More broadly, these findings show that trait data predict key properties of ecological networks, thus highlighting a role for species traits in understanding network assembly, stability, and structure.

Compositional turnover in host and parasite communities does not change network structure

Decreasing similarity between ecological communities with increasing geographic distance (i.e. distance‐decay) is a common biogeographical observation in free‐living communities, and a slightly less common observation for parasite communities. Ecological networks of interacting species may adhere to a similar pattern of decreasing interaction similarity with increasing geographic distance, especially if species interactions are maintained across space. We extend this further, examining if host–parasite networks – independent of host and parasite species identities – become more structurally dissimilar with increasing geographic distance. Utilizing a global database of helminth parasite occurrence records, we find evidence for distance‐decay relationships in host and parasite communities at both regional and global scales, but fail to detect similar relationships in network structural similarity. Host and parasite community similarity were strongly related, and both decayed rapidly with increasing geographic distance, typically resulting in complete dissimilarity after approximately 2500 km. Our failure to detect a decay in network structural similarity suggests the possibility that different host and parasite species are filling the same functional roles in interaction networks, or that variation in network similarity may be better explained by other geographic variables or aspects of host and parasite ecology.

Spatial variation in the parasite communities and genomic structure of urban rats in New York City.

Brown rats (Rattus norvegicus) are a globally distributed pest. Urban habitats can support large infestations of rats, posing a potential risk to public health from the parasites and pathogens they carry. Despite the potential influence of rodent-borne zoonotic diseases on human health, it is unclear how urban habitats affect the structure and transmission dynamics of ectoparasite and microbial communities (all referred to as "parasites" hereafter) among rat colonies. In this study, we use ecological data on parasites and genomic sequencing of their rat hosts to examine associations between spatial proximity, genetic relatedness and the parasite communities associated with 133 rats at five sites in sections of New York City with persistent rat infestations. We build on previous work showing that rats in New York carry a wide variety of parasites and report that these communities differ significantly among sites, even across small geographical distances. Ectoparasite community similarity was positively associated with geographical proximity; however, there was no general association between distance and microbial communities of rats. Sites with greater overall parasite diversity also had rats with greater infection levels and parasite species richness. Parasite community similarity among sites was not linked to genetic relatedness of rats, suggesting that these communities are not associated with genetic similarity among host individuals or host dispersal among sites. Discriminant analysis identified site-specific associations of several parasite species, suggesting that the presence of some species within parasite communities may allow researchers to determine the sites of origin for newly sampled rats. The results of our study help clarify the roles that colony structure and geographical proximity play in determining the ecology of R.norvegicus as a significant urban reservoir of zoonotic diseases. Our study also highlights the spatial variation present in urban rat parasite communities, indicating that rats across New York City are not reservoirs for a homogenous set of parasites and pathogens. As a result, the epidemiological risks may be similarly heterogeneous for people in urban habitats.

Parasite community similarity in Athabasca River trout-perch (Percopsis omiscomaycus) varies with local-scale land use and sediment hydrocarbons, but not distance or linear gradients.

Parasite communities have been shown to be structured by processes at scales ranging from continental to microhabitat, but few studies have simultaneously considered spatial and environmental variables, measured at different scales, to assess their relative influences on parasite abundance, species richness, and community similarity. Parasite abundance, diversity, and community similarity in Athabasca River trout-perch (Percopsis omiscomaycus) were examined in relation to water quality, substrate profile, metal and organic compound levels in water and sediment, and landscape use patterns at different scales, as well as distance among sites and upstream-downstream position along the river. Although species richness did not differ among sites, there were significant differences in abundance of individual taxa and community structure. We observed a shift from communities dominated by larval trematodes Diplostomum spp. to domination by gill monogeneans Urocleidus baldwini, followed by a reversion further downstream. Variations in the abundance of these taxa and of overall community similarity were strongly correlated with sediment hydrocarbons (alkanes and polycyclic aromatic hydrocarbons (PAHs)) as well as landscape use within 5km of study sites. No correlations were noted with any other predictors, indicating that parasite populations and communities in this system were likely primarily influenced by habitat level and landscape-scale filters, rather than larger-scale processes such as distance decay or river continuum effects.

Biodiversity of parasite assemblages in the genus Petrogale and its relation to the phylogeny and biogeography of their hosts

Parasites form an integral part of overall biodiversity although they are often overlooked in conservation management, where emphasis is primarily directed towards the host. Parasites are often highly specialised to particular hosts, and thus may be just as threatened as the host they inhabit. For many of Australia’s wildlife species, little is known about their associated parasite communities. To begin to address this knowledge gap, we documented the parasite fauna described in the genetically diverse marsupial genus Petrogale, which contains seven species of conservation concern. The literature evaluation showed parasites of Petrogale to be highly diverse, with 17 species of protozoa, 8 species of cestodes, 102 species of nematodes and 30 species of ectoparasites identified in 16 of 17 Petrogale host species. A comparison of the parasite communities amongst Petrogale host species indicated a highly significant correlation between the parasite community similarity, and the phylogeny (P = 0.008) and biogeography (P = 0.0001) of their Petrogale hosts, suggesting high host specificity within their associated parasite assemblages. Five Petrogale species have established species recovery programs and their parasite communities should also be considered threatened, and management of parasite diversity required as part of these conservation programs.

Assessing the influence of geographic distance in parasite communities of an exotic lizard.

The decay of similarity between biological communities with increasing geographical distance is a well-established pattern in ecology, but there are more complex factors acting on host population connections that influence this association for parasite communities, such as parasites' colonization ability and degree of connectivity between host populations. Here we aim to determine the helminth communities associated with different populations of the host lizard Hemidactylus mabouia, testing if the similarity of parasite communities decreases as the distance between them increases. For this, we collected samples of lizard populations in seven sites from Northeastern coast of Brazil and identified parasite species of helminths and pentastomids in each host, calculated the Sørensen indices of presence/absence and abundance of each pair of communities and related them to the geographical distance. We did not find a relationship of decaying similarity with increasing distance between the parasite communities of the host populations. This can be explained by factors such as the characteristics of the contact between the host populations, and by modes of transmission of most parasite species. Furthermore, it may be related to the exotic nature of the host in Brazil so that parasite communities have not reached equilibrium.

Temporal effects on host-parasite associations in four naturalized goby species living in sympatry

Introduced host species are often characterised by reduced parasite numbers compared to their native populations. Any such advantage gained from parasite release following introduction into a new area may often diminish over a short period as the new host gradually acquires local parasites. In this study, the metazoan parasite communities of four goby species (Proterorhinus semilunaris, Ponticola kessleri, Neogobius melanostomus, and Neogobius fluviatilis) recently introduced into the lower River Rhine were investigated. Mean parasite abundance and infracommunity richness were positively associated with time since host introduction, both parasite variables being the highest in P. semilunaris. In Ponticola and Neogobius species, parasite species richness and the dominance of larval parasites in the Lower Rhine were similar to that for non-native populations in the middle Danube. Sporadic local parasite acquisition and infection, predominantly by species commonly found in the native range, led to a relatively high qualitative similarity in parasite communities between hosts. The relationship between parasite abundance and fish size reflected size-dependant food selectivity and/or parasite accumulation throughout the host’s life. Data from this study emphasise the importance of duration of co-occurrence, host habitat and foraging preference, as well as the co-introduction of suitable intermediate hosts, for parasite community composition in related species.

Predicting the similarity of parasite communities in freshwater fishes using the phylogeny, ecology and proximity of hosts

Parasite communities tend to be dissimilar in hosts that are geographically, phylogenetically, ecologically and developmentally distant from one another. The decay of community similarity is a powerful and increasingly common method of studying parasite beta diversity, but most studies have examined only a single type of distance. Here, we evaluate distances based on the phylogeny, ecology, spatial proximity and size of hosts, as predictors of the similarity of parasite communities in individual hosts, host populations and host species. We surveyed parasites in six species of fish collected simultaneously from six localities in the St. Lawrence River, Canada, and species in a common group of larval parasites were discriminated using DNA sequences from barcode region of cytochrome c oxidase I. Distances based on the habitat use patterns of host species were good predictors of short‐term, ecological similarity of parasite communities, such as that operating at the scale of the individual host. The genetic distance between host species was associated with almost all types of similarity at all scales, particularly qualitative and phylogenetic similarity of parasite communities at the level of populations and meta‐populations of hosts. The trophic level, diet, spatial proximity and size of hosts were poor predictors of parasite community similarity. The increased taxonomic resolution provided by molecular data increased the explanatory power of regression models, and different factors were implicated when parasite species were distinguished with DNA barcodes than when larval parasites were lumped into morphospecies, as is commonly practiced.

The decay of parasite community similarity in ring‐billed gulls Larus delawarensis and other hosts

The similarity of parasite communities often decays with increasing geographic distance. Here, geographic distance decay is evaluated in parasite communities of 145 ring‐billed gulls from six localities in Quebec, Canada, among both individual gulls and gull populations. Spatial decay in similarity is compared to temporal decay, using host age differences as a distance measure. The similarity of parasite communities of individual gulls from localities spanning 300 km within Quebec is more strongly associated with host age differences than with geographic distance. Among gull populations in Quebec, only geographic distance is significantly related to parasite community similarity. The explanatory power of geographic distance is higher still when data from Quebec are combined with those from a study conducted 3000 km away, indicating an effect of spatial scale on geographic distance decay. The rate at which parasite community similarity decays with distance in gulls is compared with that of 17 other host species using data from published studies. Spatial scale explains two thirds of the variance in distance decay rates, and some of the remaining variance is explained by latitude. The rate of decay in parasite community similarity with distance is greater in larger scale studies and further away from the equator. Traits of host species traits such as vagility, body weight and trophic level have little or no effect on distance decay rates.

The comparative ecology and biogeography of parasites

Comparative ecology uses interspecific relationships among traits, while accounting for the phylogenetic non-independence of species, to uncover general evolutionary processes. Applied to biogeographic questions, it can be a powerful tool to explain the spatial distribution of organisms. Here, we review how comparative methods can elucidate biogeographic patterns and processes, using analyses of distributional data on parasites (fleas and helminths) as case studies. Methods exist to detect phylogenetic signals, i.e. the degree of phylogenetic dependence of a given character, and either to control for these signals in statistical analyses of interspecific data, or to measure their contribution to variance. Parasite-host interactions present a special case, as a given trait may be a parasite trait, a host trait or a property of the coevolved association rather than of one participant only. For some analyses, it is therefore necessary to correct simultaneously for both parasite phylogeny and host phylogeny, or to evaluate which has the greatest influence on trait expression. Using comparative approaches, we show that two fundamental properties of parasites, their niche breadth, i.e. host specificity, and the nature of their life cycle, can explain interspecific and latitudinal variation in the sizes of their geographical ranges, or rates of distance decay in the similarity of parasite communities. These findings illustrate the ways in which phylogenetically based comparative methods can contribute to biogeographic research.

Salinity gradient shapes distance decay of similarity among parasite communities in three marine fishes

Published data were used to compare the distance decay of similarity in parasite communities of three marine fish hosts: Atlantic cod Gadus morhua, the dab Limanda limanda and the flounder Platichthys flesus in two adjacent areas that differ with respect to the strength of a salinity gradient. In the Baltic Sea, which exhibits a strong salinity gradient from its connection with the North Sea in the west to its head in the north-east, parasite communities in all three fish hosts showed a significant decline of similarity with increasing distance. In contrast, among host populations in the North Sea, which is a fully marine environment, there was no such decline or only a weak relationship. The results suggest that environmental gradients like salinity can be strong driving forces behind patterns of distance decay in parasite communities of fishes.

Host ontogeny and the temporal decay of similarity in parasite communities of marine fish

Geographical distances between host populations are key determinants of how many parasite species they share. In principle, decay in similarity should also occur with increasing distance along any other dimension that characterizes some form of separation between communities. Here, we apply the biogeographical concept of distance decay in similarity to ontogenetic changes in the metazoan parasite communities of three species of marine fish from the Atlantic coast of South America. Using differences in body length between all possible pairs of size classes as measures of ontogenetic distances, we find that, using an index of similarity (Bray-Curtis) that takes into account the abundance of each parasite species, the similarity in parasite communities showed a very clear decay pattern; using an index (Jaccard) based on presence/absence of species only, we obtained slightly weaker but nevertheless similar patterns. As we predicted, the slope of the decay relationship was significantly steeper in the fish Cynoscion guatucupa, which goes through clear ontogenetic changes in diet and therefore in exposure to parasites, than in the other species, Engraulis anchoita and Micropogonias furnieri, which maintain a roughly similar diet throughout their lives. In addition, we found that for any given ontogenetic distance, i.e. for a given length difference between two size classes, the similarity in parasite communities was almost always higher if they were adult size classes, and almost always lower if they were juvenile size classes. This, combined with comparisons among individual fish within size classes, shows that parasite communities in juvenile fish are variable and subject to stochastic effects. We propose the distance decay approach as a rigorous and quantitative method to measure rates of community change as a function of host age, and for comparisons across host species to elucidate the role of host ecology in the development of parasite assemblages.

Similarity in parasite communities of the teleost fish Pinguipes brasilianus in the southwestern Atlantic: Infracommunities as a tool to detect geographical patterns

Patterns of distance decay in similarity among communities of the fish Pinguipes brasilianus (Teleostei: Pinguipedidae) from five areas in the southwestern Atlantic were investigated to determine whether the rate of decay varied depending on the community level or the parasite guild analyzed (ectoparasites, adult endoparasites and larval endoparasites). Similarities in species composition were computed at both the component community and infracommunity levels. Similarity indices were calculated between all possible pairs of assemblages from different zones. Infracommunity similarity values between and within host populations were averaged. Significance of linear regressions for similarity values against distance was assessed using randomization tests. Different patterns were observed for each guild, and similarity among infracommunities within host populations varied accordingly. Decay in similarity over distance was recorded for most communities. The slopes differed significantly between infracommunities and component communities in all cases, and stronger decay was always observed for infracommunities. Different geographical patterns in parasite communities were a consequence of variability in parasite availability in the different regions, modulated by oceanographic conditions, as well as variation among species in terms of host specificity and life-cycles strategies. Infracommunities showed a stronger effect of distance than component communities, probably due to the influence of short term and local variability of oceanographic conditions.

Between-population Similarity in Intestinal Parasite Community Structure of Pike (Esox Lucius)—Effects of Distance and Historical Connections

The effect of geographical distance on similarity in parasite communities of freshwater fish has received considerable attention in recent years, and it has become evident that these apparently simple relationships are influenced by, among other things, colonization ability of parasites and degree of connectivity between the populations. In the present paper, we explored qualitative and quantitative similarity in the intestinal parasite communities of pike (Esox lucius) in a particular system where previously interconnected groups of lakes became isolated ca. 8,400 yr ago. Contrary to our expectations, we did not find differences in similarity between the lake groups or a negative effect of distance among the populations. This supports the role of common ancestral colonization events and shows that no significant loss of species has occurred during the past 8,000 yr. However, the communities were dominated by a single parasite species, the cestode Triaenophorus nodulosus. The exclusion of this species from the data had a significant negative impact on the community similarities and also revealed a negative relationship between distance and quantitative similarity. This suggests that patterns of community organization may be obscured by a single dominant species. We also highlight the need for further studies in different systems and host species, as well as detailed reanalysis of existing data sets, to unravel the controversy in the relationship between distance and similarity in parasite communities.

Metazoan parasites of freshwater cyprinid fish (Leuciscus cephalus): testing biogeographical hypotheses of species diversity

The diversity and similarity of parasite communities is a result of many determinants widely considered in parasite ecology. In this study, the metazoan parasite communities of 15 chub populations (Leuciscus cephalus) were sampled across a wide geographical range. Three hypotheses of biogeographical gradients in species diversity were tested: (1) latitudinal gradient, (2) a 'favourable centre' versus 'local oasis' model, and (3) decay of similarity with distance. We found that the localities in marginal zones of chub distribution showed lower parasite species richness and diversity. A latitudinal gradient, with increasing abundance of larvae of Diplostomum species, was observed. There was a general trend for a negative relationship between relative prevalence or abundance and the distance from the locality with maximum prevalence or abundance for the majority of parasite species. However, statistical support for a 'favourable centre' model was found only for total abundance of Monogenea and for larvae of Diplostomum species. The phylogenetic relatedness of host populations inferred an important role when the 'favourable centre' model was tested. Testing of the hypothesis of 'decay of similarity with geographical distance' showed that phylogenetic distance was more important as a determinant of similarity in parasite communities than geographical distance between host populations.