RESUMO
Helminth parasites are part of almost every ecosystem, with more than 300 000 species worldwide. Helminth infection dynamics are expected to be altered by climate change, but predicting future changes is difficult owing to lacking thermal sensitivity data for greater than 99.9% of helminth species. Here, we compiled the largest dataset to date on helminth temperature sensitivities and used the Metabolic Theory of Ecology to estimate activation energies (AEs) for parasite developmental rates. The median AE for 129 thermal performance curves was 0.67, similar to non-parasitic animals. Although exceptions existed, related species tended to have similar thermal sensitivities, suggesting some helminth taxa are inherently more affected by rising temperatures than others. Developmental rates were more temperature-sensitive for species from colder habitats than those from warmer habitats, and more temperature sensitive for species in terrestrial than aquatic habitats. AEs did not depend on whether helminth life stages were free-living or within hosts, whether the species infected plants or animals, or whether the species had an endotherm host in its life cycle. The phylogenetic conservatism of AE may facilitate predicting how temperature change affects the development of helminth species for which empirical data are lacking or difficult to obtain.
Assuntos
Helmintos , Parasitos , Animais , Ecossistema , Helmintos/fisiologia , Interações Hospedeiro-Parasita/fisiologia , FilogeniaRESUMO
AbstractParasitic worms with complex life cycles have several developmental stages, with each stage creating opportunities to infect additional host species. Using a data set for 973 species of trophically transmitted acanthocephalans, cestodes, and nematodes, we confirmed that worms with longer life cycles (i.e., more successive hosts) infect a greater diversity of host species and taxa (after controlling for study effort). Generalism at the stage level was highest for middle life stages, the second and third intermediate hosts of long life cycles. By simulating life cycles in real food webs, we found that middle stages had more potential host species to infect, suggesting that opportunity constrains generalism. However, parasites usually infected fewer host species than expected from simulated cycles, suggesting that generalism has costs. There was no trade-off in generalism from one stage to the next, but worms spent less time growing and developing in stages where they infected more taxonomically diverse hosts. Our results demonstrate that life-cycle complexity favors high generalism and that host use across life stages is determined by both ecological opportunity and life-history trade-offs.
Assuntos
Acantocéfalos/fisiologia , Cestoides/fisiologia , Especificidade de Hospedeiro , Interações Hospedeiro-Parasita , Nematoides/fisiologia , Animais , Cadeia Alimentar , Estágios do Ciclo de VidaRESUMO
Parasitic worms (i.e. helminths) commonly infect multiple hosts in succession before reproducing. At each life cycle step, worms may fail to infect the next host, and this risk accumulates as life cycles include more successive hosts. Risk accumulation can be minimized by having high establishment success in the next host, but comparisons of establishment probabilities across parasite life stages are lacking. We compiled recovery rates (i.e. the proportion of parasites recovered from an administered dose) from experimental infections with acanthocephalans, cestodes and nematodes. Our data covered 127 helminth species and 16 913 exposed hosts. Recovery rates increased with life cycle progression (11%, 29% and 46% in first, second and third hosts, respectively), because larger worm larvae had higher recovery, both within and across life stages. Recovery declined in bigger hosts but less than it increased with worm size. Higher doses were used in systems with lower recovery, suggesting that high doses are chosen when few worms are expected to establish infection. Our results indicate that growing in the small and short-lived hosts at the start of a complex life cycle, though dangerous, may substantially improve parasites' chances of completing their life cycles.
Assuntos
Acantocéfalos , Helmintos , Nematoides , Animais , Interações Hospedeiro-Parasita , Estágios do Ciclo de VidaRESUMO
Parasites are important selective agents with the potential to limit gene flow between host populations by shaping local host immunocompetence. We report on a contact zone between lake and river three-spined sticklebacks (Gasterosteus aculeatus) that offers the ideal biogeographic setting to explore the role of parasite-mediated selection on reproductive isolation. A waterfall acts as a natural barrier and enforces unidirectional migration from the upstream river stickleback population to the downstream river and lake populations. We assessed population genetic structure and parasite communities over four years. In a set of controlled experimental infections, we compared parasite susceptibility of upstream and downstream fish by exposing laboratory-bred upstream river and lake fish, as well as hybrids, to two common lake parasite species: a generalist trematode parasite, Diplostomum pseudospathaceum, and a host-specific cestode, Schistocephalus solidus. We found consistent genetic differentiation between upstream and downstream populations across four sampling years, even though the downstream river consisted of ~10% first-generation migrants from the upstream population as detected by parentage analysis. Fish in the upstream population had lower genetic diversity and were strikingly devoid of macroparasites. Through experimental infections, we demonstrated that upstream fish and their hybrids had higher susceptibility to parasite infections than downstream fish. Despite this, naturally sampled upstream migrants were less infected than downstream residents. Thus, migrants coming from a parasite-free environment may enjoy an initial fitness advantage, but their descendants seem likely to suffer from higher parasite loads. Our results suggest that adaptation to distinct parasite communities can influence stickleback invasion success and may represent a barrier to gene flow, even between close and connected populations.
Assuntos
Cestoides , Infecções por Cestoides , Doenças dos Peixes , Parasitos , Smegmamorpha , Animais , Fluxo Gênico , Interações Hospedeiro-Parasita , LagosRESUMO
Compared with uninfected individuals, infected animals can exhibit altered phenotypes. The changes often appear beneficial to parasites, leading to the notion that modified host phenotypes are extended parasite phenotypes, shaped by parasite genes. However, the phenotype of a parasitized individual may reflect parasitic manipulation, host responses to infection or both, and disentangling the contribution of parasite genes versus host genes to these altered phenotypes is challenging. Using a tapeworm (Schistocephalus solidus) infecting its copepod first intermediate host, I performed a full-factorial, cross-infection experiment with five host and five parasite genotypes. I found that a behavioural trait modified by infection, copepod activity, was affected by both host and parasite genotype. There was no clear evidence for host genotype by parasite genotype interactions. Several observations indicated that host behaviour was chiefly determined by parasite genes: (i) all infected copepods, regardless of host or parasite genotype, exhibited behavioural changes, (ii) parasitism reduced the differences among copepod genotypes, and (iii) within infected copepods, parasite genotype had twice as large an effect on behaviour as host genotype. I conclude that the altered behaviour of infected copepods primarily represents an extended parasite phenotype, and I discuss how genetic variation in parasitic host manipulation could be maintained.
Assuntos
Cestoides , Copépodes , Parasitos , Animais , Genótipo , Interações Hospedeiro-ParasitaRESUMO
Parasitologists have worked out many complex life cycles over the last ~150 yr, yet there have been few efforts to synthesize this information to facilitate comparisons among taxa. Most existing host-parasite databases focus on particular host taxa, do not distinguish final from intermediate hosts, and lack parasite life-history information. We summarized the known life cycles of trophically transmitted parasitic acanthocephalans, cestodes, and nematodes. For 973 parasite species, we gathered information from the literature on the hosts infected at each stage of the parasite life cycle (8,510 host-parasite species associations), what parasite stage is in each host, and whether parasites need to infect certain hosts to complete the life cycle. We also collected life-history data for these parasites at each life cycle stage, including 2,313 development time measurements and 7,660 body size measurements. The result is the most comprehensive data summary available for these parasite taxa. In addition to identifying gaps in our knowledge of parasite life cycles, these data can be used to test hypotheses about life cycle evolution, host specificity, parasite life-history strategies, and the roles of parasites in food webs.
Assuntos
Acantocéfalos/fisiologia , Cestoides/fisiologia , Nematoides/fisiologia , Parasitos/fisiologia , Animais , Interações Hospedeiro-Parasita , Estágios do Ciclo de VidaRESUMO
Non-random species associations occur in naturally sampled parasite communities. The processes resulting in predictable community structure (e.g. particular host behaviours, cross-immunity, interspecific competition) could be affected by traits that vary within a parasite species, like growth or antigenicity. We experimentally infected three-spined sticklebacks with a large tapeworm (Schistocephalus solidus) that impacts the energy needs, foraging behaviour and immune reactions of its host. The tapeworms came from two populations, characterized by high or low growth in sticklebacks. Our goal was to evaluate how this parasite, and variation in its growth, affects the acquisition of other parasites. Fish infected with S. solidus were placed into cages in a lake to expose them to the natural parasite community. We also performed a laboratory experiment in which infected fish were exposed to a fixed dose of a common trematode parasite. In the field experiment, infection with S. solidus affected the abundance of four parasite species, relative to controls. For two of the four species, changes occurred only in fish harbouring the high-growth S. solidus; one species increased in abundance and the other decreased. These changes did not appear to be directly linked to S. solidus growth though. The parasite exhibiting elevated abundance was the same trematode used in the laboratory infection. In that experiment, we found a similar infection pattern, suggesting that S. solidus affects the physiological susceptibility of fish to this trematode. Associations between S. solidus and other parasites occur and vary in direction. However, some of these associations were contingent on the S. solidus population, suggesting that intraspecific variability can affect the assembly of parasite communities.
Assuntos
Cestoides/fisiologia , Infecções por Cestoides/veterinária , Doenças dos Peixes/parasitologia , Smegmamorpha , Trematódeos/fisiologia , Infecções por Trematódeos/veterinária , Animais , Biota , Infecções por Cestoides/imunologia , Infecções por Cestoides/parasitologia , Feminino , Doenças dos Peixes/imunologia , Interações Hospedeiro-Parasita , Masculino , Smegmamorpha/genética , Smegmamorpha/crescimento & desenvolvimento , Infecções por Trematódeos/imunologia , Infecções por Trematódeos/parasitologiaRESUMO
Complex life cycles are common in free-living and parasitic organisms alike. The adaptive decoupling hypothesis postulates that separate life cycle stages have a degree of developmental and genetic autonomy, allowing them to be independently optimized for dissimilar, competing tasks. That is, complex life cycles evolved to facilitate functional specialization. Here, I review the connections between the different stages in parasite life cycles. I first examine evolutionary connections between life stages, such as the genetic coupling of parasite performance in consecutive hosts, the interspecific correlations between traits expressed in different hosts, and the developmental and functional obstacles to stage loss. Then, I evaluate how environmental factors link life stages through carryover effects, where stressful larval conditions impact parasites even after transmission to a new host. There is evidence for both autonomy and integration across stages, so the relevant question becomes how integrated are parasite life cycles and through what mechanisms? By highlighting how genetics, development, selection and the environment can lead to interdependencies among successive life stages, I wish to promote a holistic approach to studying complex life cycle parasites and emphasize that what happens in one stage is potentially highly relevant for later stages.
Assuntos
Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , Parasitos/crescimento & desenvolvimento , Parasitos/genética , Animais , Evolução Biológica , Meio Ambiente , Interações Hospedeiro-Parasita/genética , Metamorfose BiológicaRESUMO
Helminths often occupy defined niches in the gut of their definitive hosts. In the dioecious acanthocephalans, adult males and females usually have similar gut distributions, but sexual site segregation has been reported in at least some species. We studied the intestinal distribution of the acanthocephalan Echinorhynchus borealis von Linstow, 1901 (syn. of E. cinctulus Porta, 1905) in its definitive host, burbot (Lota lota Linnaeus). Over 80% of female worms were found in the pyloric caeca, whereas the majority of males were in the anterior two-thirds of the intestine. This difference was relatively consistent between individual fish hosts. Worms from different parts of the gut did not differ in length, so site segregation was not obviously related to worm growth or age. We found proportionally more males in the caeca when a larger fraction of the females were found there, suggesting mating opportunities influence gut distribution. However, this result relied on a single parasite infrapopulation and is thus tentative. We discuss how mating strategies and/or sexual differences in life history might explain why males and females occupy different parts of the burbot gut.
RESUMO
The acanthocephalan Echinorhynchus bothniensis Zdzitowiecki and Valtonen, 1987 differs from most other species in the genus Echinorhynchus Zoega in Müller, 1776 by infecting mysids (order Mysida) instead of amphipods (order Amphipoda) as intermediate hosts. Here we report on the occurrence of E. bothniensis in mysids (Mysis segerstralei Audzijonyte et Väinölä) and in its fish definitive hosts in a high Arctic lake. Out of 15 907 sampled mysids, 4.8% were infected with a mean intensity of 1.05 worms (range 1-5), although there was notable variation between samples taken in different years and sites. Larger mysids appear more likely to be infected. Of five fish species sampled, charr,Salvelinus alpinus (Linnaeus), and a benthic-feeding whitefish morph, Coregonus lavaretus (Linnaeus), were the most heavily infected (mean abundances of 80 and 15, respectively). The adult parasite population in fish exhibited a female-biased sex ratio (1.78 : 1). Although E. bothniensis is rather unique in infecting mysids, many aspects of its natural history mirror that of other acanthocephalan species.
RESUMO
The acanthocephalan Echinorhynchus salmonis Müller, 1784 is a common parasite of salmonid fish, but it has rarely been reported from an intermediate host. Samples of benthic amphipods, Monoporeia affinis (Lindström), were taken from multiple, deep sites (usually below 70 m) in the Gulf of Bothnia over the course of more than a decade and examined for acanthocephalans. Overall, only 0.44% of 23 296 amphipods were infected, all with just a single worm. This prevalence is consistent with several previous reports of acanthocephalans in deep-water, benthic amphipods, but it appears low compared to that often reported for acanthocephalan species infecting littoral amphipods. Parasite occurrence did not exhibit a clear regional pattern (i.e. northern vs southern sites) nor did it have any relationship with site depth. At sites sampled over multiple years, parasite abundance was consistently low (mostly < 0.01), though two spikes in abundance (over 0.06) were also observed, indicating that infection can be substantially higher at particular times or in particular places. The median density of E. salmonis in samples containing the parasite was estimated as 8.4 cystacanths per m(2).
RESUMO
Parasitic worms (helminths) frequently have complex life cycles in which they are transmitted trophically between two or more successive hosts. Sexual reproduction often takes place in high trophic-level (TL) vertebrates, where parasites can grow to large sizes with high fecundity. Direct infection of high TL hosts, while advantageous, may be unachievable for parasites constrained to transmit trophically, because helminth propagules are unlikely to be ingested by large predators. Lack of niche overlap between propagule and definitive host (the trophic transmission vacuum) may explain the origin and/or maintenance of intermediate hosts, which overcome this transmission barrier. We show that nematodes infecting high TL definitive hosts tend to have more successive hosts in their life cycles. This relationship was modest, though, driven mainly by the minimum TL of hosts, suggesting that the shortest trophic chains leading to a host define the boundaries of the transmission vacuum. We also show that alternative modes of transmission, like host penetration, allow nematodes to reach high TLs without intermediate hosts. We suggest that widespread omnivory as well as parasite adaptations to increase transmission probably reduce, but do not eliminate, the barriers to the transmission of helminths through the food web.
Assuntos
Evolução Biológica , Cadeia Alimentar , Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , Nematoides/fisiologia , Animais , Ecossistema , Invertebrados/parasitologia , Nematoides/crescimento & desenvolvimento , Reprodução , Vertebrados/parasitologiaRESUMO
Schistocephalus solidus is one of the few cestodes that can be bred in vitro. Worms have typically been bred in pairs, so the parents of each offspring can clearly be assigned. From a genetic perspective, it would be useful to be able to mate an individual worm to multiple partners while still being able to distinguish among different parents. As each adult S. solidus possesses numerous reproductive complexes, cutting worms and breeding the pieces separately would facilitate such breeding designs. We halved worms before in vitro breeding and evaluated whether this affected outcrossing rates and reproductive output. Cutting did not influence clutch mass, i.e. egg number and size, or outcrossing rates, but eggs from cut worms had a lower hatching rate than eggs from uncut worms. We found that when two anterior worm halves were bred together, they produced fewer, smaller eggs with higher hatching rates, compared to two posterior halves. Moreover, once we controlled for this effect of 'worm half', the two halves of an individual worm tended to reproduce similarly under comparable circumstances. We conclude that cutting plerocercoids increases the flexibility with which this tapeworm can be experimentally bred without dramatically affecting the production of viable, outcrossed eggs.
Assuntos
Cruzamento/métodos , Cestoides/fisiologia , Infecções por Cestoides/veterinária , Doenças dos Peixes/parasitologia , Smegmamorpha/parasitologia , Análise de Variância , Animais , Infecções por Cestoides/parasitologia , Óvulo/fisiologia , ReproduçãoRESUMO
Organisms with complex life cycles occupy distinct niches as larvae and adults. One presumed advantage of this is the ability to exploit different resources successively throughout ontogeny. Various taxa, however, have evolved nonfeeding, nongrowing adult stages. We show theoretically that this counterintuitive no-growth strategy is favored when the optimal larval size is greater than or equal to the optimal adult size for reproduction. We empirically investigated this in a group of parasitic worms (helminths). Helminths are transmitted trophically between hosts before reproducing in large, high-trophic-level hosts, and most undergo considerable growth as adults in their final host. Some well-studied tapeworm species (Schistocephalus, Ligula, and Digramma species) are notable exceptions; they reproduce semelparously without any growth in their final habitat (the gut of piscivorous birds). Using cross-species comparative analyses, we show that these tapeworms that do not grow in their final host (1) attain larval sizes in their last intermediate host (fishes) that are comparable to or larger than the adult sizes reached by tapeworms that do grow in the same adult niche (also piscivorous birds) and (2) are large, even as larvae, relative to the mass of their final hosts. These results are consistent with the idea that a massive larval size can make adult growth superfluous, and we discuss whether this likely applies to other complex life cycle taxa with nonfeeding, nongrowing adults.
Assuntos
Aves/parasitologia , Cestoides/crescimento & desenvolvimento , Peixes/parasitologia , Interações Hospedeiro-Parasita , Animais , Tamanho Corporal , Cestoides/fisiologia , Feminino , Trato Gastrointestinal/parasitologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Modelos Biológicos , Reprodução , Especificidade da EspécieRESUMO
Evolutionary stasis characterizes many phenotypes, even ones that seem suboptimal. Among tapeworms, Schistocephalus solidus and its relatives have some of the shortest developmental times in their first intermediate hosts, yet their development still seems excessively long considering they can grow faster, larger, and safer in the next hosts in their complex life cycles. I conducted 4 generations of selection on the developmental rate of S. solidus in its copepod first host, pushing a conserved-but-counterintuitive phenotype toward the limit of known tapeworm life-history strategies. Faster parasite development evolved and enabled earlier infectivity to the stickleback next host, but low heritability for infectivity moderated fitness gains. Fitness losses were more pronounced for slow-developing parasite families, irrespective of selection line, because directional selection released linked genetic variation for reduced infectivity to copepods, developmental stability, and fecundity. This deleterious variation is normally suppressed, implying development is canalized and thus under stabilizing selection. Nevertheless, faster development was not costly; fast-developing genotypes did not decrease copepod survival, even under host starvation, nor did they underperform in the next hosts, suggesting parasite stages in successive hosts are genetically decoupled. I speculate that, on longer time scales, the ultimate cost of abbreviated development is reduced size-dependent infectivity.
Assuntos
Cestoides , Infecções por Cestoides , Copépodes , Doenças dos Peixes , Parasitos , Smegmamorpha , Animais , Larva , Interações Hospedeiro-Parasita , Doenças dos Peixes/parasitologia , Cestoides/genética , Infecções por Cestoides/parasitologia , Smegmamorpha/genética , Copépodes/genéticaRESUMO
Parasitic worms (helminths) with complex life cycles divide growth and development between successive hosts. Using data from 597 species of acanthocephalans, cestodes, and nematodes with two-host life cycles, we found that helminths with larger intermediate hosts were more likely to infect larger, endothermic definitive hosts, although some evolutionary shifts in definitive host mass occurred without changes in intermediate host mass. Life-history theory predicts parasites to shift growth to hosts in which they can grow rapidly and/or safely. Accordingly, helminth species grew relatively less as larvae and more as adults if they infected smaller intermediate hosts and/or larger, endothermic definitive hosts. Growing larger than expected in one host, relative to host mass/endothermy, was not associated with growing less in the other host, implying a lack of cross-host trade-offs. Rather, some helminth orders had both large larvae and large adults. Within these taxa, however, size at maturity in the definitive host was unaffected by changes to larval growth, as predicted by optimality models. Parasite life-history strategies were mostly (though not entirely) consistent with theoretical expectations, suggesting that helminths adaptively divide growth and development between the multiple hosts in their complex life cycles.
Assuntos
Helmintos , Parasitos , Animais , Evolução Biológica , Interações Hospedeiro-Parasita , Larva , Estágios do Ciclo de VidaRESUMO
Why do so many parasitic worms have complex life-cycles? A complex life-cycle has at least two hypothesized costs: (i) worms with longer life-cycles, i.e. more successive hosts, must be generalists at the species level, which might reduce lifetime survival or growth, and (ii) each required host transition adds to the risk that a worm will fail to complete its life-cycle. Comparing hundreds of trophically transmitted acanthocephalan, cestode, and nematode species with different life-cycles suggests these costs are weaker than expected. Helminths with longer cycles exhibit higher species-level generalism without impaired lifetime growth. Further, risk in complex life-cycles is mitigated by increasing establishment rates in each successive host. Two benefits of longer cycles are transmission and production. Longer cycles normally include smaller (and thus more abundant) first hosts that are likely to consume parasite propagules, as well as bigger (and longer-lived) definitive hosts, in which adult worms grow to larger and presumably more fecund reproductive sizes. Additional factors, like host immunity or dispersal, may also play a role, but are harder to address. Given the ubiquity of complex life-cycles, the benefits of incorporating or retaining hosts in a cycle must often exceed the costs.
RESUMO
Parasitic worms (i.e., helminths) commonly infect multiple hosts in succession. With every transmission step, they risk not infecting the next host and thus dying before reproducing. Given this risk, what are the benefits of complex life cycles? Using a dataset for 973 species of trophically transmitted acanthocephalans, cestodes, and nematodes, we tested whether hosts at the start of a life cycle increase transmission and whether hosts at the end of a life cycle enable growth to larger, more fecund sizes. Helminths with longer life cycles, that is, more successive hosts, infected conspicuously smaller first hosts, slightly larger final hosts, and exploited trophic links with lower predator-prey mass ratios. Smaller first hosts likely facilitate transmission because of their higher abundance and because parasite propagules were the size of their normal food. Bigger definitive hosts likely increase fecundity because parasites grew larger in big hosts, particularly endotherms. Helminths with long life cycles attained larger adult sizes through later maturation, not faster growth. Our results indicate that complex helminth life cycles are ubiquitous because growth and reproduction are highest in large, endothermic hosts that are typically only accessible via small intermediate hosts, that is, the best hosts for growth and transmission are not the same.
Assuntos
Helmintos , Nematoides , Parasitos , Animais , Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , ReproduçãoRESUMO
For organisms with a complex life cycle, a large larval size is generally beneficial, but it may come at the expense of prolonged development. Individuals that grow fast may avoid this tradeoff and switch habitats at both a larger size and younger age. A fast growth rate itself can be costly, however, as it requires greater resource intake. For parasites, fast larval growth is assumed to increase the likelihood of host death before transmission to the next host occurs. Using the tapeworm Schistocephalus solidus in its copepod first intermediate host, I investigated potential constraints in the parasite's larval life history. Fast-growing parasites developed infectivity earlier, indicating there is no functional tradeoff between size and developmental time. There was significant growth variation among full-sib worm families, but fast-growing sibships were not characterized by lower host survival or more predation-risky host behavior. Parental investment also had little effect on larval growth rates. The commonly assumed constraints on larval growth and development were not observed in this system, so it remains unclear what prevents worms from exploiting their intermediate hosts more aggressively.
Assuntos
Evolução Biológica , Cestoides/crescimento & desenvolvimento , Interações Hospedeiro-Parasita , Larva/crescimento & desenvolvimento , Animais , Comportamento AnimalRESUMO
Trophically transmitted parasites often alter their intermediate host's phenotype, thereby predisposing the hosts to increased predation. This is generally considered a parasite strategy evolved to enhance transmission to the next hosts. However, the adaptive value of host manipulation is not clear as it may be associated with costs, such as increased susceptibility to predators that are unsuitable next hosts for the parasites. We examined the ratio between the benefits and costs of host manipulation for transmission success of Acanthocephalus lucii (Acanthocephala), a parasite that alters the hiding behaviour and pigmentation of its isopod hosts. We experimentally compared the susceptibility of infected and uninfected isopods to predation by perch (Perca fluvialis; definitive host of the parasite) and dragonfly larvae (dead end). We found that the parasite predisposed the isopods to predation by both predators. However, the increased predation vulnerability of the infected isopods was higher towards perch. This suggests that, despite the costs due to non-host predation, host manipulation may still be advantageous for the parasite.