In silico characterization of cysteine-stabilized αß defensins from neglected unicellular microeukaryotes.

BACKGROUND: The emergence of multi-resistant pathogens have increased dramatically in recent years, becoming a major public-health concern. Among other promising antimicrobial molecules with potential to assist in this worldwide struggle, cysteine-stabilized αß (CS-αß) defensins are attracting attention due their efficacy, stability, and broad spectrum against viruses, bacteria, fungi, and protists, including many known human pathogens. RESULTS: Here, 23 genomes of ciliated protists were screened and two CS-αß defensins with a likely antifungal activity were identified and characterized, using bioinformatics, from a culturable freshwater species, Laurentiella sp. (LsAMP-1 and LsAMP-2). Although any potential cellular ligand could be predicted for LsAMP-2; evidences from structural, molecular dynamics, and docking analyses suggest that LsAMP-1 may form stably associations with phosphatidylinositol 4,5-bisphosphates (PIP2), a phospholipid found on many eukaryotic cells, which could, in turn, represent an anchorage mechanism within plasma membrane of targeted cells. CONCLUSION: These data stress that more biotechnology-oriented studies should be conducted on neglected protists, such ciliates, which could become valuable sources of novel bioactive molecules for therapeutic uses.

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.

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.

In vitro toxicity of isolated strains and cyanobacterial bloom biomasses over Paramecium caudatum (ciliophora): Lessons from a non-metazoan model organism.

Cyanobacteria have been considered a major global threat because of their widespread ability to proliferate and contaminate inland and marine waters with toxic metabolites. For this reason, to avoid risks to humans and environmental health, regulatory legislation and guidelines have been established based on extensive toxicological data. However, most of what is known in this field come from works on microcystin (MC) variants, which effects were almost exclusively tested in metazoan models. In this work, we used acute end-point toxicological assays and high-resolution hybrid quadrupole time-of-flight mass spectrometer coupled with electrospray ionization source (ESI-Q-TOF-MS) analyses to evaluate the deleterious impact of aqueous extracts prepared from cultures of cyanobacteria and environmental bloom biomasses over a non-metazoan model organism, the cosmopolitan fresh/brackish water unicellular microeukaryote, Paramecium caudatum (Ciliophora). Our data suggest that all extracts produced time-dependent effects on P. caudatum survival, irrespective of their metabolite profile; and that this ciliate is more sensitive to extracts containing microginins than to extracts with only MCs, stressing that more toxicological investigations should be performed on the environmental impact of neglected cyanotoxins. Further, our data provide evidence that P. caudatum may be more sensitive to cyanotoxins than vertebrates, indicating that guidelines values, set on metazoans are likely to be inaccurate to protect organisms from basal food web positions. Thus, we highly recommend the widespread use of microeukaryotes, such as ciliates in environmental risk assessment frameworks for the establishment of more reliable cyanotoxin monitoring guideline values.

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.

Ciliates as model organisms for the ecotoxicological risk assessment of heavy metals: A meta-analysis.

Ciliates are key components of aquatic ecosystems, significantly contributing to the decomposition of organic matter and energy transfer to higher trophic levels. They are considered good biological indicators of chemical pollution and relatively sensitive to heavy metal contamination. In this study, we performed a meta-analysis of the available toxicity data of heavy metals and ciliates to assess: (1) the sensitivity of freshwater ciliates to different heavy metals, (2) the relative sensitivity of ciliates in comparison to the standard test species used in ecotoxicological risk assessment, and (3) the difference in sensitivity across ciliate taxa. Our study shows that the tolerance of ciliates to heavy metals varies notably, which is partly influenced by differences in methodological conditions across studies. Ciliates are, in general, sensitive to Mercury > Cadmium > Copper > Zinc > Lead > Chromium. Also, this study shows that most ciliates are more tolerant to heavy metal pollution than the standard test species used in ecotoxicological risk assessments, i.e., Raphidocelis subcapitata, Daphnia magna, and Onchornyncus mykiss. Threshold concentrations derived from toxicity data for these species is expected to confer sufficient protection for the vast majority of ciliate species. Our data analysis also shows that the most commonly tested ciliate species, Paramecium caudatum and Tetrahymena thermophila, are not necessarily the most sensitive ones to heavy metal pollution. Finally, this study stresses the importance of developing standard toxicity test protocols for ciliates, which could lead to a better comprehension of the toxicological impact of heavy metals and other contaminants to ciliate species.

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.

Molecular characterization of Theileria equi in horses from the state of Rio de Janeiro, Brazil.

Theileria equi is one of the etiologic agents of the equine piroplasmosis. This infectious disease is transmitted by ticks and is a worldwide problem in the international horse movement. The 18S rRNA gene of T. equi is often used for genotyping and phylogenetic purpose. This study aimed to analyze the degree of the heterogeneity of the 18S rRNA gene of T. equi in horses from the state of Rio de Janeiro, Brazil. The complete T. equi 18S rRNA sequences were obtained from twenty naturally infected horses. The PCR amplicons were cloned and sequenced. The phylogenetic analyses were performed using a set of T. equi 18S rRNA sequences and other related organisms available in ARB-Silva database. There were twelve distinct T. equi 18S rRNA gene sequences circulating in horses in the state of Rio de Janeiro, Brazil. Monophyletic clades with 2% evolutionary divergence between clades and high bootstrap value were the support to divide T. equi sequences in three distinct clades. The sequences from this study grouped into clades I (70%, n=14/20) and II (30%, n=6/20). All of the T. equi sequences grouped within a node other than the theileriids. This study reported a clear division of two distinct genotypes of T. equi 18S rRNA sequences in state of Rio de Janeiro, Brazil, and it demonstrates that distinct isolates of T. equi can coexist in the same geographic region.

Multidisciplinary re-description of Plasmodium (Novyella) paranucleophilum in Brazilian wild birds of the Atlantic Forest kept in captivity.

Haemosporidian blood parasites of the Plasmodium genus are the causative agents of avian malaria in many parts of the world. Despite the great diversity of Brazilian avifauna, few studies have been conducted to examine the haemosporidians of wild birds found in the Brazilian Atlantic Forest, especially those kept in captivity. This study aimed to re-examine and further characterize the South American avian parasite Plasmodium paranucleophilum using a multidisciplinary approach. Blood samples were collected from 68 captive birds representing 15 species found in the Atlantic Forest of southeastern Brazil. Morphometric and morphological characterization was performed, in addition to PCR and sequencing of the mitochondrial cytochrome b gene and subsequent phylogenetic analysis. The overall prevalence of P. paranucleophilum infection in the study was 13.23% (n = 9), with a mean parasitemia of 0.58%. We observed the highest parasitemia of 3.88% in Rupornis magnirostris. In our phylogenetic analysis, P. paranucleophilum and P lasmodium nucleophilum formed distinct, highly supported clades, with a mean genetic divergence of 2.48%. This study provides new morphological and molecular data, expanding our knowledge of the haemosporidians of wild birds in Brazil and highlighting the need for further investigation. The true depth of diversity in Brazilian avian haemosporidians remains largely unknown, and given the enormous variety of vectors and avian species, there may be many more species of these blood parasites yet to be described.

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.

Morphology and 18S rDNA gene sequence of Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea) from Brazil.

The morphology and morphometric data of seven populations of Blepharisma sinuosum from southeastern Brazil were investigated. The description is based on live observations, protargol impregnation, and scanning electron microscopy. Blepharisma sinuosum measures 75-255µm in length and 25-93µm in width and has a spindle-shaped body, pink color, a single contractile vacuole located at the posterior end, 50 adoral membranelles, a conspicuous paroral, 17-35 somatic kineties, a moniliform macronucleus with 2-7 connected nodules, and 3-20 micronuclei. Morphological comparisons with similar species were performed and suggest that B. americanum is the junior synonym of B. sinuosum. The 18S rDNA gene sequence of B. sinuosum was obtained and compared with that of other Blepharisma species. The length and GC content of the obtained sequence is 1652bp and 47.03%, respectively, and has a very high structural similarity (99.9%) with the B. undulans sequence. The validity of the classification of Blepharisma species in morphonuclear subgenera is also discussed.