Are sexes equally parasitized in damselflies and dragonflies?

Parasitism plays an essential part in ecology and evolution of host species and understanding the reasons for differential parasitism within and among hosts species is therefore important. Among the very important factors potentially affecting parasitism is the gender of the host. Here, we studied whether either females or males are more likely to harbour parasites among Odonatan insects, by relying on an extensive literature review and new field data. We collected data on numerous dragonfly and damselfly species and their ectoparasites (water mites) and endoparasites (gregarines) to examine the generality of similarities and differences in prevalence, intensity and maximum number of parasites of male and female hosts. We found three main results. Firstly, most of the odonate host species showed no differences between sexes in either gregarine or water mite prevalence and intensity. The only exception was female damselflies’ higher gregarine prevalence and intensity compared to conspecific males. These inequalities in gregarine parasitism may be due to behavioral and physiological differences between conspecific males and females. In comparison, there were no differences in dragonflies between sexes in water mite or gregarine prevalence and intensity. Secondly, damselflies had higher prevalence and intensity levels of both gregarine and water mite parasites compared to dragonflies. Finally, we found a strong species level pattern between female and male parasitism: a certain level of gregarine or water mite parasitism in one sex was matched with a similar parasitism level for the other. This indicates similar exposure and susceptibility to parasites on both sexes. Even though significant differences of parasite levels between the sexes were observed within certain host species, our results strongly suggest that on a general level a more parasitized sex does not exist in the order, Odonata.     

Odonates,gregarines and water mites: why are the same host species infected by both parasites?

1. Damselflies and dragonflies are widely parasitised insects and numerous studies have tried to understand this host–parasite relationship. However, most of these studies have concentrated on a single host species, neglecting the larger pattern within the Odonata order. 2. The aim of this paper was to examine different damselfly and dragonfly species for common endo‐ and ectoparasites and whether a general infection pattern can be found. Additionally, the goal was to investigate whether the phylogeny of the host species could explain these possible infection patterns. To this end, a dataset from the existing literature was compiled and the prevalence of endoparasitic gregarines and ectoparasitic water mites was analysed for 46 different odonate species. 3. Three distinct patterns were found: (i) most of the odonate host species had both gregarines and water mites, rather than only either one or neither; (ii) there appears to be a positive association between gregarine and water mite prevalences across host species; (iii) a weak phylogenetic signal was detected in gregarine prevalence and a strong one in water mite prevalence. 4. It is hypothesised that, due to the infection and transmission mechanisms by which water mites and gregarines infect different odonate host species, parasitism is aggregated to common, high‐density species. However, much research is needed in order to fully understand this relationship between odonates and their parasites, especially within the same host populations and host species assemblages.     

Two or more enteropathogens are associated with diarrhoea in Mexican children

Background

The Apicomplexa are a diverse group of obligate protozoan parasites infesting a wide range of invertebrate and vertebrate hosts including humans. These parasites are notoriously difficult to control and many species continue to evolve resistance to commercial antibiotics. In this study, we sought to find an effective chemotherapeutic treatment against arthropod gregarines (Apicomplexa), and to identify candidate compounds for testing against other groups of protozoan parasites.

Methods

We tested eleven commercial antibiotics against a gregarine parasite of Romalea microptera grasshoppers. Infected insects were fed daily, lettuce containing known amounts of specific antibiotics. On Days 15 or 20, we measured the number of gregarines remaining in the digestive tract of each grasshopper.

Results

Treatment with metronidazole and griseofulvin in host insects significantly reduced gregarine counts, whereas, gregarine counts of insects fed, albendazole, ampicillin, chloramphenicol, fumagillin, quinine, streptomycin, sulfadimethoxine, thiabendazole or tetracycline, were not significantly different from the controls. However, albendazole produced a strong, but non-significant reduction in gregarine count, and streptomycin exhibited a non-significant antagonistic trend.

Conclusion

Our results confirm that gregarine infections are difficult to control and suggest the possibility that streptomycin might aggravate gregarine infection. In addition, the insect system described here, provides a simple, inexpensive, and effective method for screening antibiotics.     

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

A parasite's modification of host behavior reduces predation on its host

Parasite modification of host behavior is common, and the literature is dominated by demonstrations of enhanced predation on parasitized prey resulting in transmission of parasites to their next host. We present a case in which predation on parasitized prey is reduced. Despite theoretical modeling suggesting that this phenomenon should be common, it has been reported in only a few host–parasite–predator systems. Using a system of gregarine endosymbionts in host mosquitoes, we designed experiments to compare the vulnerability of parasitized and unparasitized mosquito larvae to predation by obligate predatory mosquito larvae and then compared behavioral features known to change in the presence of predatory cues. We exposed Aedes triseriatus larvae to the parasite Ascogregarina barretti and the predator Toxohrynchites rutilus and assessed larval mortality rate under each treatment condition. Further, we assessed behavioral differences in larvae due to infection and predation stimuli by recording larvae and scoring behaviors and positions within microcosms. Infection with gregarines reduced cohort mortality in the presence of the predator, but the parasite did not affect mortality alone. Further, infection by parasites altered behavior such that infected hosts thrashed less frequently than uninfected hosts and were found more frequently on or in a refuge within the microcosm. By reducing predation on their host, gregarines may be acting as mutualists in the presence of predation on their hosts. These results illustrate a higher‐order interaction, in which a relationship between a species pair (host–endosymbiont or predator–prey) is altered by the presence of a third species.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Record of a gregarine (Apicomplexa: Neogregarinida) in the abdomen of the termite Coptotermes gestroi (Isoptera, Rhinotermitidae)

Coptotermes gestroi is an exotic species of termite that is a pest of great economical importance in Brazil. This paper relates the occurrence of a coelomic gregarine (Apicomplexa: Neogregarinida) in the abdomen of the foraging workers recently collected from field colonies of this termite. The termite hosts presented large, white abdomens because they carried 1 up to 3 cysts of gregarines filled with numerous lemon-shaped spores. Earlier developmental stages of this gregarine were not observed in the scanning microscope preparations nor in the histological slides of the infected termites. However, the lemon-shaped spores suggest a parasite gregarine of Mattesia genus, family Lipotrophidae.     

Higher gregarine parasitism often in sibling species of host damselflies with smaller geographical distributions

1. This study investigated inter‐specific variation in parasitism by gregarines (Eugregarinorida: Actinocephalidae), among sibling species of damselflies (Odonata: Zygoptera), in relation to relative size of geographical ranges of host species. 2. Gregarines are considered generalist parasites, particularly for taxonomically related host species collected at the same sites or area. Prevalence and median intensity of gregarine parasitism was obtained for 1338 adult damselflies, representing 14 species (7 sibling species pairs) across 3 families within the suborder Zygoptera. Damselflies were collected at three local sites in Southeastern Ontario, during the same periods over the season. 3. Five out of seven species pairs had significant differences in parasitism between sibling species. The less widespread host species was the more parasitised for three species pairs with significant differences in gregarine prevalence, and for two species pairs with differences in median intensity. The more widespread host had a higher intensity of infection as expected, in two species pairs. 4. Future studies on ecological determinants of parasitism among related species should examine robust measures of abundance of species and representation of species regionally.     

Electrophoretic differences in the isoenzymes of two mosquito gregarines, Ascogregarina barretti and A. geniculati

Cross-infection experiments demonstrated that Ascogregarina barretti, from Aedes triseriatus, completes its life cycle in Aedes geniculatus. Parasite numbers were comparable to infection with Ascogregarina geniculati, making the separation of these parasites by host preference difficult. However, electrophoresis readily distinguished isoenzymes from the two morphologically similar gregarine species. Different migration rates were obtained for isocitrate dehydrogenase 1 and 2, lactate dehydrogenase, and malate dehydrogenase. The migration rates were also different for parasite and host isoenzymes. When a single, heavily infected gut was subjected to electrophoresis the isocitrate dehydrogenase bands of each were clearly distinguishable on the same electrophoretic track. Electrophoresis appears to be a reliable method for resolving taxonomic complications of mosquito gregarines, a group often with wide host specificities and variable taxonomic characters.     

Influence of gut parasites on growth performance in the water strider Gerris buenoi (Hemiptera: Gerridae)

Water striders harbor a diverse assemblage of symbionts in their digestive tract We used a field experiment, in which water striders were reared in enclosures in their natural pond habitat, to assess the effects of gut symbionts on growth Trypanosomatid flagellates had significant adverse effects on both development time and adult size, and therefore are clearly parasitic Yet because of their low prevalences (2% or less), trypanosomatids cannot be a major factor in the dynamics of our study population Gregannes occurred in 36% of the water striders, often in high numbers, and filling the entire midgut of some bugs Nevertheless, infected and uninfected gerrids did not differ in their growth, and gregarine loads were uncorrelated with development time and adult size attained We also did not find effects of gregarines in a second experiment with different rearing conditions, including a treatment with food stress We used a quantitative genetic approach to test if resistance against gregarines has a heritable component There was no evidence for any genetic variation, suggesting that variability in gregarine loads is the result of environmental heterogeneity Comparison with published reports from water striders shows that there is a great variability in the diversity and prevalence of symbionts among different populations and species, of gerrids     

Parasitism affects worker size in the Neotropical swarm-founding social wasp, <Emphasis Type="Italic">Polybia paulista</Emphasis> (Hymenoptera,Vespidae)

Summary We discovered two kinds of parasites, i.e., a strepsipteran, possibly Xenos myrapetrus (Trois) and an undescribed gregarine in the Neotropical swarm-founding paper wasp, Polybia paulista (Ihering). Although proportions of workers that were infected by these parasites varied greatly among colonies analyzed, prevalence of infected workers was recognized. Five external characters were measured and compared among uninfected workers, stylopized (i.e., infected by Strepsiptera) workers and workers that were infected by gregarines. Uninfected workers were significantly larger than stylopized workers, while smaller than workers that were infected by gregarines. Nutrients of stylopized workers may be plundered during their growth period, and consequently their body size may be reduced. However, the gregarines may manipulate host larvae to solicit more food from adults or increase development time of larvae longer, and therefore produce more parasites from a larger host.Received 1 July 2003; revised 1 December 2003; accepted 4 December 2003.     

Description of Ganymedes yurii sp. n. (Ganymedidae), a New Gregarine Species from the Antarctic Amphipod Gondogeneia sp. (Crustacea)

A novel species of aseptate eugregarine, Ganymedes yurii sp. n., is described using microscopic and molecular approaches. It inhabits the intestine of Gondogeneia sp., a benthic amphipod found along the shore of James Ross Island, Weddell Sea, Antarctica. The prevalence of the infection was very low and only a few caudo‐frontal syzygies were found. Morphologically, the new species is close to a previously described amphipod gregarine, Ganymedes themistos, albeit with several dissimilarities in the structure of the contact zone between syzygy partners, as well as other characteristics. Phylogenetic analysis of the 18S rDNA from G. yurii supported a close relationship between these species. These two species were grouped with other gregarines isolated from crustaceans hosts (Cephaloidophoroidea); however, statistical support throughout the clade of Cephaloidophoroidea gregarines was minimal using the available dataset.     

Why the –omic future of Apicomplexa should include gregarines

Gregarines, a polyphyletic group of apicomplexan parasites infecting mostly non-vertebrates hosts, remains poorly known at taxonomic, phylogenetic and genomic levels. However, it represents an essential group for understanding evolutionary history and adaptive capacities of apicomplexan parasites to the remarkable diversity of their hosts. Because they have a mostly extracellular lifestyle, gregarines have developed other cellular developmental forms and host–parasite interactions, compared with their much better studied apicomplexan cousins, intracellular parasites of vertebrates (Hemosporidia, Coccidia, Cryptosporidia). This review highlights the promises offered by the molecular exploration of gregarines, that have been until now left on the side of the road of the comparative –omic exploration of apicomplexan parasites. Elucidating molecular bases for both their ultrastructural, functional and behavioural similarities and differences, compared with those of the typical apicomplexan models, is expected to provide entirely novel clues on the adaptive capacities developed by Apicomplexa over evolution. A challenge remains to identify which gregarines should be explored in priority, as recent metadata from open and host-associated environments have confirmed how underestimated is our current view on true gregarine biodiversity. It is now time to turn to gregarines to widen the currently highly skewed view we have of adaptive mechanisms developed by Apicomplexa.     

Acephaline gregarine parasites (Monocystis sp.) are not transmitted sexually among their lumbricid earthworm hosts

The precise transmission mode(s) of acephaline gregarines in their earthworm hosts has long been questioned, yet a rigorous experimental evaluation of sexual transmission is currently lacking. That Monocystis sp., a common gregarine parasite of the earthworm Lumbricus terrestris, infects the sexual organs of its host is suggestive of sexual transmission. Considering the divergent evolutionary consequences of various modes of transmission, excluding or proving sexual transmission in this host-parasite system is critical to fully understanding it. We cultured uninfected earthworms from cocoons and subsequently mated them to either an infected or uninfected partner (from the wild). We then compared these individuals with an orally infected group, which were infected using a newly developed gavage (oral injection) method. Our data have unambiguously established that (1) horizontal sexual transmission does not play a significant role in the transmission of Monocystis sp., and (2) oral transmission through the soil is likely the principal mode of transmission between earthworms. This finding is important to models of mate-choice because infection avoidance does not appear to drive mating decisions. Finally, we further report a successful and relatively simple method to obtain infection-free individuals, which can subsequently be infected via oral gavage and used in empirical studies.     

Comparative surface morphology of marine coelomic gregarines (Apicomplexa, Urosporidae): Pterospora floridiensis and Pterospora schizosoma

Two species in the aseptate gregarine genus Pterospora from the Pacific and Gulf coasts were analyzed by scanning electron microscopy, which revealed characteristics not reported in other gregarines. The gamonts of these species had branching trunks that ended in terminal digits, and both species moved by cytoplasmic streaming and peristalsis. Pterospora floridiensis had surface pits and tracts of parallel ridges that bended and connected with one another. Pterospora schizosoma had irregular-shaped surface swellings that were usually arranged in rosette patterns. These unique surface features have not been reported for other gregarines, and are strikingly different from the surface features of many septate and aseptate gregarines that inhabit the intestinal lumena of their hosts and move by gliding. The correlation of Pterospora's unique pellicular features to the habitat and cytoplasmic streaming characteristic of the genus may be significant, and may reflect an adaptation for development in coelomic environments.     

Morphology and phylogenetic position of two novel marine gregarines (Apicomplexa,Eugregarinorida) from the intestines of North‐eastern Pacific ascidians

Eugregarine apicomplexans parasitize marine, freshwater and terrestrial invertebrates, and have lifecycles involving trophozoites (feeding stages) with complex morphologies and behaviour. The genus Lankesteria refers to marine aseptate eugregarines that parasitize ascidians. We described the surface ultrastructure of two new gregarine species, L. chelyosomae sp. n. and L. cystodytae sp. n. that inhabit the intestines of Chelyosoma columbianum and Cystodytes lobatus, respectively, collected from the North‐eastern Pacific Ocean. Apart from inhabiting different hosts and major differences in the cell size of L. chelyosomae sp. n. (mean length 182 µm) and L. cystodytae sp. n. (mean length 70 µm), the morphology of both gregarine species was quite similar. The trophozoites ranged from elliptoid to obdeltoid in shape and were brownish in colour. The nucleus was situated at the anterior end of the cell just behind a pointed mucron. A dense array of epicytic knobs was present over the entire surface of trophozoites in both species, and longitudinal epicytical folds were only weakly developed. We also sequenced the small subunit rDNA from the gregarines collected from both hosts, which supported the establishment of two new Lankesteria species. Phylogenetic analyses of the new DNA sequences and those derived from other alveolates, demonstrated that both new species clustered in a strongly supported clade consisting of other Lankesteria species, Lecudina species, and some environmental sequences. These morphological and molecular phylogenetic data suggested that improved knowledge of gregarine diversity could lead to the recognition of more than one distinct clade (genus) of gregarines within ascidian hosts.     

Molecular Phylogenetic Positions of Two New Marine Gregarines (Apicomplexa)—Paralecudina anankea n. sp. and Lecudina caspera n. sp.—from the Intestine of Lumbrineris inflata (Polychaeta) Show Patterns of Co‐evolution

Gregarine apicomplexans are unicellular parasites commonly found in the intestines and coeloms of invertebrate hosts. Traits associated with the conspicuous feeding stage of gregarines, known as the trophozoite, have been used in combination with molecular phylogenetic data for species delimitation and the reconstruction of evolutionary history. Trophozoite morphology alone is often inadequate for inferring phylogenetic relationships and delimiting species due to frequent cases of high intraspecific variation combined with relatively low interspecific variation. The current study combined morphological data with small subunit (SSU) rDNA sequences to describe and establish two novel marine gregarine species isolated from the intestine of a polychaete host Lumbrineris inflata collected in British Columbia (Canada): Paralecudina anankea n. sp. and Lecudina caspera n. sp. The sister species to the host is Lumbrineris japonica, which can be found on the opposite side of the Pacific Ocean (Japan) and contains two different species of gregarine parasites: Paralecudina polymorpha and Lecudina longissima. Molecular phylogenetic analyses placed P. anankea n. sp. as the sister species to P. polymorpha and L. caspera n. sp. as the sister species to L. longissima. This phylogenetic pattern demonstrates a co‐evolutionary history whereby speciation of the host (Lumbrineris) corresponds with simultaneous speciation of the two different lineages of intestinal gregarines (Paralecudina and Lecudina).     

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae)

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.     

Differential identification of Ascogregarina species (Apicomplexa: Lecudinidae) in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by polymerase chain reaction

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straight-forward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.