Rumen ciliates (Ciliophora, Trichostomatia) in Brazilian domestic cattle feeding on diets with crescent urea levels.

Since their discovery, rumen ciliates of domestic cattle have been reported from various geographic locations. However, until now there is only one taxonomic inventory of ciliates associated with Brazilian cattle. The present study aimed to assess the community composition, relative abundance, richness, and density of rumen ciliates in Brazilian cattle, whose feeding diets were supplemented with crescent urea levels. Across all treatments analyzed, one subclass, two orders, three families, 11 genera, and 31 species of ciliates were identified. The ciliate community composition and species richness varied among the four treatments used. However, the total ciliate density was not affected by the experimental diets. We described a new entodiniid morphotype, Entodinium caudatum m. orbonuclearis, and recorded Oligoisotricha bubali for the second time in Brazil.

Insights on the taxonomy of Haemoproteus parasites infecting cracid birds.

The Haemosporida order is a well-supported clade of heteroxenous parasites transmitted by dipteran insects and frequently found parasitizing wild birds. These parasites have already been reported in all zoogeographic regions of the world, except for Antarctica. One of the potential hosts of haemosporidians is the Cracidae family, which includes approximately 50 species, 22 of which are present in Brazil, classified within nine genera. Data on haemosporidian infecting individuals of the Cracidae family is scarce, with only three Haemoproteus species being recorded in this group of birds. We found Haemoproteus spp. infection in all Penelope obscura bronzina analyzed. Among the parasites found, we observed two lineages of Haemoproteus (PENOBS02 and PENOBS03), which were characterized by morphological, molecular and phylogenetic approaches. The morphological data on cracid haemosporidian parasites, together with our phylogenetic results, allows discussions on the taxonomy of the Haemoproteus parasites that infect birds of the Cracidae family.

Rumen ciliates (Alveolata, Ciliophora) associated with goats: checklist, geographic distribution, host specificity, phylogeny and molecular dating.

Although the diversity (~35 species) and worldwide distribution of goats (Ruminantia, Bovidae, Caprinae) are significant, studies on the diversity of symbiont ciliates in these mammals are scarce in comparison to other ruminants. The present work is a review and checklist of species based on taxonomic, morphologic, and ecologic studies of rumen ciliate protozoa in goats, presenting geographic distribution and hosts, as well as estimating the macroevolutionary relationships of the species observed in the studies. To that end, all of the available literature on databases was reviewed, the schematic drawings were made based on information present in the original description of the taxa, and the phylogenetic relationships were inferred based on Maximum Likelihood and Bayesian Inference analyses. According to our review, 72 species and 14 genera of ciliates have been associated with goats. Through the analysis of the association between ciliate genera and caprine hosts, it was shown that ciliates are more associated with domestic animals (Capra hircus14 genera) than wild ones (Rupicapra rupicaprasix genera, Capra ibexone genus, Capra pyrenaicaone genus). Thirteen countries were identified in the distribution map as having had reports of ciliate species associated with goats. The interaction networks of ciliates and their hosts showed that the species of ciliates associated with goats also occur in other herbivore mammal species. The recovered phylogenetic hypotheses show that the ciliate species in goats form a non-monophyletic group with maximum and minimum ages of ~8.2My and ~2.4My. We have also found that a large portion of the studies on the diversity of ciliates in goats does not employ all necessary techniques in an integrative way, despite it being essential for detailed descriptions and better knowledge of this fraction of biodiversity.

Insights into the systematics of the family Ophryoscolecidae (Ciliophora, Entodiniomorphida).

The family Ophryoscolecidae currently comprises 225 species of trichostomatid ciliates, subdivided into three subfamilies (Entodiniinae, Diplodiniinae, and Ophryoscolecinae). The last taxonomic review of the family was performed 55 years ago, but recent morphological and molecular studies indicate the need for a profound review of the systematics of this taxon, since its current taxonomy is insufficient to organize the diversity of the group. Here, we briefly review the systematics of the family Ophryoscolecidae based on information recovered from the literature and new morphological and molecular data. We add four new 18S rDNA sequences of ophryoscolecids to molecular databases, which contributed to improving the comprehension of intrafamily relationships within this group. Finally, we discuss some systematic problems and suggest approaches to resolve such inconsistencies in the future.

Systematic review of the genus Ostracodinium (Ciliophora, Entodiniomorphida, Ophryoscolecidae) and notes on the taxonomy of Ostracodinium rugoloricatum Kofoid amp; MacLennan, 1932.

The family Ophryoscolecidae (Ciliophora, Entodiniomorphida) constitutes a diverse and monophyletic group of symbiotic ciliates of herbivorous mammals. The family includes approximately 200 species, distributed in three subfamilies and sixteen genera. The subfamily Diplodiniinae is the most diverse group in Ophryoscolecidae and comprises the genus Ostracodinium, which includes species with two retractable ciliary zones in the anterior body portion, a broad skeletal plate covering almost all the right surface of the body and a variable number of contractile vacuoles. The genus currently comprises 28 species, classified according to body size and shape, position and shape of the nuclear apparatus, number and position of contractile vacuoles, and number and shape of caudal projections. The present study performs a systematic review of the genus Ostracodinium, based on morphological and molecular data, and provides data about geographic distribution and hosts of each species.

New Finds on the Systematics of Cycloposthiid Ciliates (Ciliophora: Entodiniomorphida: Cycloposthiidae) Based on New 18S-rDNA Sequences from a Brazilian Capybara.

The family Cycloposthiidae (Entodiniomorphida) comprises ciliated protists that are symbionts of the gastrointestinal tract of several herbivore mammals, such as rodents, elephants, equids, primates, hippopotamus, marsupials, rhinoceros and tapir, where they contribute to the digestion of their host's plant-based diet. Despite the significance of these ciliates to the evolution of their hosts, many characters used in the taxonomy of the group are homoplastic and most of the valid species do not have molecular data available. For these reasons the systematics of this family is poorly understood. Here, we sequenced the 18S-rDNA of ten cycloposthiids, including nine Cycloposthium spp. and Monoposthium cynodontum, all of them isolated from the cecum of a Brazilian capybara. Our phylogenetic analyses indicate that the family Cycloposthiidae might be polyphyletic, while M. cynodontum and Cycloposthium spp. constitute a single monophyletic group. Given the great morphological and molecular similarities between members of M. cynodontum and Cycloposthium ciliates, it is possible that this species, although it has been described in the genus Monoposthium, is actually a Cycloposthium ciliate.

Rediscovery of Plasmodium (Huffia) huffi (Apicomplexa, Haemosporida): a lost lineage from toucans.

Literature data on toucans haemosporidians are scarce and all reports come from investigations in Brazil. Muniz et al. (Rev Bras Malariol 3: 339-356, Muniz et al., Rev Bras Malariol 3:339-356, 1951) and Muniz and Soares (Rev Bras Malar 611-617, Muniz J, Soares R de RL (1954) Nota sôbre um parasita do gênero Plasmodium encontrado no Ramphastos toco Müller, 1776, "Tucano-Açu", e diferente do Plasmodium huffi: Plasmodium pinottii n. sp. Rev Bras Malar 611 - 617.) described two Plasmodium species, P. huffi and P. pinottii, in Ramphastos toco. Later, Manwell and Sessler (J Protozol 18: 570-574, Manwell and Sessler, Malaria Parasites of Toucans J Protozol 18:570-574, 1971) established a new subspecies, P. nucleophilum toucani. In the last review on avian haemosporidians, Valkiunas (Valkiunas, Avian malaria parasites and other haemosporidia, CRC Press, New York, 2005) highlighted that P. huffi was insufficiently characterized, considering it a lost lineage. Also, the original description of P. huffi was considered insufficiently clear, due to a possible co-infection of the toucan hosts with a Novyella-like species. Here, we redescribed the species Plasmodium (Huffia) huffi based on morphological and molecular data, which were found in two toucan species, Ramphastos toco and Pteroglossus aracari from Brazil. Morphological features of the specimens are markedly the same as the original description. In R. toco, we observe two individuals infected, one infected only with P. huffi and one co-infected with P. huffi and the Novyella-like species, as observed in the original description. Also, we observe one R. toco infected only with the Novyella-like species, identified by morphological and molecular data as Plasmodium (Novyella) nucleophilum nucleophilum. In this way, it was possible to redescribe Plasmodium huffi in detail, without the doubt characters observed in the original description. Moreover, by applying species delimitation algorithms to a large Plasmodium phylogeny, we were able to identify new possible hosts for P. huffi and extend its geographic distribution to include North America.

New morphological and molecular data for Haemoproteus (H.) paramultipigmentatus in the Atlantic Forest of Brazil.

Parasites of the genus Haemoproteus have been reported in almost all avian clades and zoogeographic regions, except Antarctica. However, despite the large number of reports worldwide, they are poorly studied in the Neotropical region, which includes the Atlantic Forest, a biodiversity hotspot with more than 890 bird species, of which 24% are endemic. Haemoproteus (Haemoproteus) paramultipigmentatus was described by morphological and molecular analyses in 2013 infecting Columbiform birds in Mexico. However, since the original description this parasite has not been studied in detail. Here, we investigate the prevalence of Haemoproteus spp. in Brazilian Columbiformes and conducted a taxonomic integrative study of the species Haemoproteus (Haemoproteus) paramultipigmentatus, including new morphological and molecular data from a Brazilian population. Moreover, we provide discussions about the geographic distribution and phylogenetic relationships between different lineages of this parasite. Our findings demonstrated a high prevalence of Haemoproteus spp. infection in Brazilian Columbiformes, which is in accordance with previous studies. Morphological characterization of H. paramultipigmentatus revealed minor differences from the original description. Through molecular and phylogenetic analyses we identified a new lineage of H. paramultipigmentatus that was added to the genetic databases. Our findings also suggest a new geographical distribution for this hemoparasite, including South American countries, and raise discussions about its current distribution.

Checklist of Cycloposthiidae species (Ciliophora, Entodiniomorphida), with a brief review on taxonomy, morphology and hosts.

The family Cycloposthiidae include 69 species, distributed in 17 genera, all of them described in symbiotic association with various mammalian host species. The taxonomic classification of the family is confused because of its broad diagnosis and absence of synapomorphies. To validate taxa and eliminate synonyms, the present work performed a brief taxonomic review of Cycloposthiidae, compiling morphological characterization, host records, and geographic location data. According to our review, the family should be instead, composed of 13 genera and 66 species; it was observed that the currently in use taxonomic characters do not reflect evolutionary divergence; and that the broad host range of Cycloposthiidae may be linked to cortex characteristics of these organisms or because of the fact that they do not constitute a monophyletic group. Thus, the performance of more molecular phylogenetic studies, including more cycloposthiid representatives and as well as the use of recent morphological techniques would be necessary to clarify the taxonomy of the group.

Morphology, molecular phylogeny, and taxonomy of trichodinids (Ciliophora, Mobilida) from Calanoid copepods.

Trichodinid ciliates were isolated from calanoid copepods and were characterized by morphological and molecular techniques. The trichodinids found in this study were morphologically similar to Trichodina acuta, but morphometric differences were observed between our specimens and T. acuta. The T. acuta specimens, isolated from fish in several geographical locations around the world, were slightly larger than specimens in our population. In fact, our morphological and morphometric data are more similar to Trichodina diaptomi populations, also found on copepods. Moreover, our phylogenetic analyses revealed that the 18S rRNA gene sequence from our samples emerged in a clade with two T. acuta sequences, infesting fish hosts in China. These three sequences showed 100% identity. Our data, along with a taxonomic review, suggest that the species T. diaptomi, described recently, is actually a synonym of T. acuta and that this species may be related to fish and copepod hosts. In conclusion, we highlight the urgent need to better investigate the polymorphism and host specificity within the Mobilida, as this data may represent important characters to better understand the evolution into this order.

Helmet-shaped Body of Entodiniomorphid Ciliates (Ciliophora, Entodiniomorphida), a Synapomorphy or a Homoplasy?

Triadinium was created to include Triadinium caudatum. Further, four other species were included, T. minimum, T. galea, T. elongatum, and T. magnum, all sharing a characteristic helmet-shaped body. Wolska and Grain argued that the inclusion of T. minimum and T. galea into Triadinium was done based on superficial morphological aspects, and established two new genera to accommodate these species: Circodinium and Gassovskiella. Although the phylogenetic relationships within Entodiniomorphida have been investigated by multiple authors, none of them discussed the evolutionary relationship of helmet-shaped entodiniomorphids. We performed molecular phylogenetics and revisited old literature digging for morphological data to explain our results. According to our analyses, the helmet-shaped body is homoplastic and may have evolved from at least three different entodiniomorphid ancestors. Circodinium minimum is phylogenetically related to members of Blepharocorythidae, T. caudatum emerged within Spirodiniidae and G. galea within Polydiniellidae. This phylogenetic hypothesis is partially supported by information on infraciliature and ultrastructure of C. minimum and T. caudatum. However, such morphological information is not available for polydiniellids. In order to shed some light into the evolution of the helmet-shaped ciliates, future works should focus to collect information on the infraciliature and the ultrastructure of Polydiniella mysorea and of other Triadinium species.

Morphological and Morphometric Characterization of Eodinium posterovesiculatum (Ciliophora, Ophryoscolecidae) in Brazilian Cattle

Abstract The morphological variability of Eodinium posterovesiculatum (Ciliophora, Trichostomatia) has been interpreted in different ways: four distinct species or four morphotypes of the same species. The present study aims to perform morphological and morphometric comparative analysis of the four morphotypes found in cattle from the southeastern region of Brazil. Ruminal content samples were obtained from four Holstein x Gir cattle and fixed at 18.5% formalin for morphological analysis. Morphometry was performed based on individuals stained with Lugol's solution [1]. The infraciliary bands were stained using silver carbonate impregnation technique [2]. Morphological and morphometric characterizations, supported by literature, suggest that the four morphotypes of E. posterovesiculatum are actually a single polymorphic species due to small morphometric differences and mostly identical morphological characters, with the format of the caudal processes being the only morphological characteristic that sets them apart.

Intestinal ciliates (Alveolata, Ciliophora) in Brazilian domestic horses (Equus caballus L.) and a review on the ciliate communities associated with horses around the world.

The aims of this study were to identify and quantify the gastrointestinal ciliates from Brazilian domestic horses, to compare the data obtained with previous studies and to review data on ciliate communities associated with horses around the world. We used eight domestic horses, kept under extensive management. Fecal samples were obtained immediately after defecation of animals and fixed in 18.5% formaldehyde. We identify 24 species of endosymbiotic ciliates, distributed in two subclasses, two orders, five families and 13 genera. The ciliate composition was similar among the animals analyzed. The family Blepharocorythidae presented the higher prevalence and the family Buetschliidae was observed in greater abundance and density. Among the species identified, Cycloposthium dentiferum was observed for the first time in Central and South America and Bundleia elongata, B. inflata, B. piriformis, B. vorax and Cycloposthium psicauda, were observed for the first time in the American continent. In addition, the species Buissonella tapiri, which was originally discovered in fecal contents of the American tapir (Tapirus terrestris) in Brazil was observed for the first time associated to Equus caballus. The review on the ciliate communities associated with horses around the world demonstrated that the equine ciliates are a very diversified group with representatives in two subclasses, Suctoria and Trichostomatia, totaling 78 species distributed in three orders and six families, having been registered in different equine hosts (Equus asinus, Equus caballus and Equus quagga) and geographic locations (USA, China, Russia, Taiwan, Mexico, Cyprus, Turkey, Kyrgyzstan and Brazil).

Polymorphism evidence in Plasmodium (Haemamoeba) lutzi Lucena, 1939 (Apicomplexa, Haemosporida) isolated from Brazilian wild birds.

Plasmodium parasites can infect great variety of bird species around the world inflicting the so called avian malaria, an illness that could be fatal in some cases and consequently, should be monitored and widely included into conservation programs. The aim of this study was to characterize two lineages of Plasmodium (Haemamoeba) lutzi found in some birds in the Atlantic Forest of Minas Gerais - Brazil, that were morphologically identified after blood smears analyses under light microscopy and molecularly by sequencing the mitochondrial cytochrome b gene (cyt b). Besides these two lineages could be clearly morphologically identified as P.(H.) lutzi, some variations in comparison with its original description were noticed: absence of meronts and gametocytes (early and fully grown) in polychromatic erythrocytes, the larger size of pigment granules in meronts and gametocytes, and the presence of small vacuoles between pigment accumulation in fully grow macrogametocytes. Moreover, a certain degree of genetic intraspecific diversity was also observed across the lineages of P. (H.) lutzi, indicating the existence of polymorphisms within this taxon, which is uncommon in Haemosporida. These results allow discussion about species boundaries within avian hemosporidians and highlight the importance of multidisciplinary approaches for a more efficient species identification and characterization.

Trichostomatid Ciliates (Alveolata, Ciliophora, Trichostomatia) Systematics and Diversity: Past, Present, and Future.

The gastrointestinal tracts of most herbivorous mammals are colonized by symbiotic ciliates of the subclass Trichostomatia, which form a well-supported monophyletic group, currently composed by ∼1,000 species, 129 genera, and 21 families, distributed into three orders, Entodiniomorphida, Macropodiniida, and Vestibuliferida. In recent years, trichostomatid ciliates have been playing a part in many relevant functional studies, such as those focusing in host feeding efficiency optimization and those investigating their role in the gastrointestinal methanogenesis, as many trichostomatids are known to establish endosymbiotic associations with methanogenic Archaea. However, the systematics of trichostomatids presents many inconsistencies. Here, we stress the importance of more taxonomic works, to improve classification schemes of this group of organisms, preparing the ground to proper development of such relevant applied works. We will present a historical review of the systematics of the subclass Trichostomatia highlighting taxonomic problems and inconsistencies. Further on, we will discuss possible solutions to these issues and propose future directions to leverage our comprehension about taxonomy and evolution of these symbiotic microeukaryotes.

Morphology and description of infraciliary bands pattern in four Metadinium Awerinzew Mutafowa, 1914 species (Ciliophora, Entodiniomorphida, Ophryoscolecidae) with taxonomic notes on the genus.

The genus Metadinium includes ophryoscolecid ciliates, with two retractile ciliary zones in the anterior body portion, two broad skeletal plates, a lobed macronucleus and two contractile vacuoles. Species belonging to this genus were recorded in several geographical locations and in different host species. However, they were mostly described based on a single ciliatological technique, and until now, there is only one description of the infraciliary pattern in Metadinium ciliates (Metadinium medium). The present study performs the morphological and infraciliature characterization of four species belonging to Metadinium: M. esalqum, M. minorum, M. rotundatum and M. ypsilon; and presents taxonomic notes on the genus. The oral infraciliature in these four Metadinium species corresponds to the Diplodinium-type, pattern shared with genera Diplodinium, Diploplastron, Eudiplodinium, Eremoplastron and Polyplastron. A brief review conducted highlights the difficulty of delimiting Metadinium species due to the morphological similarity among them.

Description of Diploplastron dehorityi sp. nov. (Entodiniomorphida, Ophryoscolecidae), a new rumen ciliate from Brazilian sheep (Ovis aries).

To date the genus Diploplastron comprised only one species of ophryoscolecid ciliate, Diploplastron affine, which is characterized by having two retractable ciliary zones in the anterior end of the body, two slender and juxtaposed skeletal plates on the right side, a rod shape macronucleus, and two contractile vacuoles. During study on the characterization of rumen ciliate community composition in Brazilian domestic sheep, we observed ciliates with atypical morphology but with diagnostic features of genus Diploplastron. This study describes Diploplastron dehorityi, a new species of ophryoscolecid ciliate, that differs from D. affine, primarily, in the morphology of skeletal plates, morphology of nuclear apparatus and body shape. In addition to the similarities between the new species and congener species, D. dehorityi has some morphological similarities to species of genus Eremoplastron.

Sheep fed with banana leaf hay reduce ruminal protozoa population.

A ciliate protozoa suppression can reduce methane production increasing the energy efficiency utilization by ruminants. The physicochemical characteristics of rumen fluid and the profile of the rumen protozoa populations were evaluated for sheep fed banana leaf hay in replacement of the Cynodon dactylon cv. vaqueiro hay. A total of 30 male sheep were raised in intensive system during 15 days of adaptation and 63 days of experimental period. The animals were distributed in a completely randomized design that included six replicates of five treatments with replacement levels (0, 25, 50, 75, and 100%) of the grass vaquero for the banana leaf hay. Samples of fluid were collected directly from the rumen with sterile catheters. Color, odor, viscosity, and the methylene blue reduction potential (MBRP) were evaluated and pH estimated using a digital potentiometer. After decimal dilutions, counts of genus protozoa were performed in Sedgewick Rafter chambers. The averages of pH, MBRP, color, odor, and viscosity were not influenced by the inclusion of the banana leaf hay. However, the total number of protozoa and Entodinium spp. population significantly decreased at 75 and 100% inclusions of banana leaf hay as roughage.

Phylogenetic Analyses Support Validity of Genus Eodinium (Ciliophora, Entodiniomorphida, Ophryoscolecidae).

The validity of genus Eodinium has been historically disputed due to morphological similarities with Diplodinium (absence of skeletal plates as well as adoral and dorsal ciliary zones at the same body level). To address this issue, the 18S rDNA of four Eodinium posterovesiculatum morphotypes and four Diplodinium anisacanthum morphotypes were sequenced and phylogenetically analyzed. The different inference methods suggest the existence of a last common ancestor of Eodinium and Ostracodinium that is not shared with Diplodinium, strongly supporting the validity of genus Eodinium. Since skeletal plates are present in all members of genus Ostracodinium, the most parsimonious is a secondary loss of skeletal plates in E. posterovesiculatum. This work represents a breakthrough in the taxonomy and phylogeny of the family Ophryoscolecidae indicating that the skeletal plates may not reflect evolutionary divergence within this group of ciliates as traditionally proposed.

Rumen ciliates in Brazilian sheep (Ovis aries), with new records and redescription of Entodinium contractum (Entodiniomorphida: Ophryoscolecidae).

The species composition, prevalence, and average relative abundance of rumen ciliates were analyzed in 16 Brazilian sheep. 28 species of ciliates were identified belonging to 3 families: Isotrichidae, Ophryoscolecidae, and Parentodiniidae. Among these ciliates, Entodinium alces, Metadinium esalqum, and M. rotundatum were found for the first time in sheep, and other 12 species for the first time in Brazilian sheep. Different morphotypes of parentodiniid ciliates were identified in 7 of the 16 sheep analyzed and this is the second report of this family in ruminants. The species Entodinium contractum was redescribed based on the new data on the general morphological features; for the first time, we described the oral infraciliature, which in this species is of the Entodinium-type. In this species, it is noteworthy that the prominent vestibular polybrachykinety can be used as an important identifying characteristic.