Tubulin-mediated anatomical and functional changes caused by Ca2+ in human erythrocytes

In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton. (AU)

Exploring the protist microbiome: The diversity of bacterial communities associated with Arcella spp. (Tubulina: Amoebozoa).

Research on protist-bacteria interactions is increasingly relevant as these associations are now known to play important roles in ecosystem and human health. Free-living amoebae are abundant in all environments and are frequent hosts for bacterial endosymbionts including pathogenic bacteria. However, to date, only a small fraction of these symbionts have been identified, while the structure and composition of the total symbiotic bacterial communities still remains largely unknown. Here, we use the testate amoeba Arcella spp. as model organisms to investigate the specificity and diversity of Arcella-associated microbial communities. High-throughputamplicon sequencing from the V4 region of the 16S rRNA gene revealed high diversity in the bacterial communities associated with the wild Arcella spp. To investigate the specificity of the associated bacterial community with greater precision, we investigated the bacterial communities of two lab-cultured Arcella species, A. hemispherica and A. intermedia, grown in two different media types. Our results suggest that Arcella-bacteria associations are species-specific, and that the associated bacterial community of lab-cultured Arcella spp. remains distinct from that of the surrounding media. Further, each host Arcella species could be distinguished based on its bacterial composition. Our findings provide insight into the understanding of eukaryotic-bacterial symbiosis.

Gill amoebae from freshwater rainbow trout (Oncorhynchus mykiss): In vitro evaluation of antiparasitic compounds against Vannella sp.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose-response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml).

High Diversity of Testate Amoebae (Amoebozoa, Arcellinida) Detected by HTS Analyses in a New England Fen using Newly Designed Taxon-specific Primers.

Testate (shell-building) amoebae, such as the Arcellinida (Amoebozoa), are useful bioindicators for climate change. Though past work has relied on morphological analyses to characterize Arcellinida diversity, genetic analyses revealed the presence of multiple cryptic species underlying morphospecies. Here, we design and deploy Arcellinida-specific primers for the SSU-rDNA gene to assess the community composition on the molecular level in a pilot study of two samplings from a New England fen: (1) 36-cm horizontal transects and vertical cores; and (2) 26-m horizontal transects fractioned into four size classes (2-10, 10-35, 35-100, and 100-300 µm). Analyses of these data show the following: (1) a considerable genetic diversity within Arcellinida, much of which comes from morphospecies lacking sequences on GenBank; (2) communities characterized by DNA (i.e. active + quiescent) are distinct from those characterized by RNA (i.e. active, indicator of biomass); (3) active communities on the surface tend to be more similar to one another than to core communities, despite considerable heterogeneity; and (4) analyses of communities fractioned by size find some lineages (OTUs) that are abundant in disjunct size categories, suggesting the possibility of life-history stages. Together, these data demonstrate the potential of these primers to elucidate the diversity of Arcellinida communities in diverse habitats.

Postoperative infection caused by Acinetobacter baumannii misdiagnosed as a free-living amoeba species in a humeral head hemiarthroplasty patient: a case report.

BACKGROUND: Acinetobacter baumannii is ubiquitous, facultative intracellular, and opportunistic bacterial pathogen. Its unique abilities allow it to survive in a diverse range of environments, including health care settings, leading to nosocomial infections. And its exceptional ability to develop resistance to multiple antibiotics leaves few drug options for treatment. It has been recognized as a leading cause of nosocomial pneumonia and bacteremia over the world. CASE PRESENTATION: In this case, a 73-year-old woman presented with a Neer Group VI proximal humeral fracture. Six hours after a successfully performed hemiarthroplasty, she developed continuous fever. Clinical examination revealed that the vitals were regular. Laboratory and radiographic examinations revealed only elevated procalcitonin levels. Blood culture revealed no bacterial or fungal growth. Cooling treatment and empirical broad-spectrum antibiotic therapy showed no apparent effect. CONCLUSIONS: We report a postoperative infection caused by Acinetobacter baumannii. The infectious pathogen was identified via molecular DNA sequencing and was initially misidentified as a free-living amoeba species upon microscopic examinations. The patient was mistreated with antiamebic combination therapy. Her symptoms persisted for over 4 months and were eventually followed by her death.

Molecular identification of Vermamoeba vermiformis from freshwater fish in lake Taal, Philippines.

Free Living Amoebae (FLA) are considered ubiquitous. FLAs may infect various biological organisms which act as reservoir hosts. Infected freshwater fishes can pose a public health concern due to possible human consumption. This study aims to identify possible pathogenic FLAs present in freshwater fishes. Seventy five (75) Oreochromis niloticus were studied for the presence of FLAs. Fish organs were suspended in physiologic saline pelleted and cultured in non-nutrient agar (NNA) lawned with Escherichia coli and were incubated in 33 °C for 14 days. Eighteen (18) fish gills and nineteen (19) fish intestine samples presented with positive growth. Trophozoites and cystic stages of FLAs were subcultured until homogenous growth was achieved. Cells were harvested from cultured plates and DNA was extracted using Chelex resin. DNA was subjected to polymerase chain reaction using universal forward primer EukA and reverse primer EukB targeting the 18s RNA. Of the 37 plates that presented with positive amoebic growth, 9 samples showed the presence of DNAs and were sent for further purification and sequencing. Basic Local Alignment Search Tool (BLAST) results showed that protists isolated from fish organs in Lake Taal include: Eocercomonas (HM536152), Colpoda steinii (KJ607915) and Vermamoeba vermiformis (KC161965). The results showed that fresh-water fishes can harbour FLAs in the gut. It is proposed that freshwater reservoirs utilized for aquaculture be monitored for the presence of FLAs and extensive study be conducted on the pathogenicity of bacterial endosymbionts and infecting viruses to its mammalian and non-mammalian host.

Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida).

We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

Amoebic gill infection in coho salmon Oncorhynchus kisutch farmed in Korea.

About 70% mortality occurred in cultured coho salmon Oncorhynchus kisutch at a marine farm in the South Sea of Korea in 2014. Diseased fish showed greyish or pale patches on the gills, with no internal signs of disease. No bacteria or viruses were isolated from diseased fish, but numerous amoebae were found on the gills. Histopathological examinations revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae had a 630 bp partial 18S rRNA gene fragment specific to Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that this Korean amoeba belonged to the N. perurans group. This is the first report of N. perurans infection in Korea.

New lipid-dependent Malassezia species from parrots / Nuevas especies lipodependientes del género Malassezia procedentes de loros

Background. All the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals. Aim. In this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov. Methods. The isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed. Results. The strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species. Conclusions. These studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots (AU)

Antecedentes. Todas las especies del género Malassezia actualmente identificadas se han aislado de mamíferos. Sin embargo, tan solo unas pocas de ellas se han aislado de aves. De hecho, las aves han sido estudiadas con menos frecuencia como portadoras de estas levaduras que los mamíferos. Objetivos. En este estudio describimos dos nuevas especies del género Malassezia: Malassezia brasiliensis sp. nov. y Malassezia psittaci sp. nov. Métodos. Las cepas estudiadas en esta publicación se aislaron de loros utilizados como animales de compañía en Brasil. Las cepas se caracterizaron mediante los criterios morfológicos y fisiológicos actualmente utilizados para la identificación de estas levaduras. También se llevó a cabo la secuenciación y el análisis de los fragmentos génicos D1/D2 26S e ITS-5.8S del ADN ribosómico y del gen de la β-tubulina. Resultados. Los perfiles fenotípicos de las cepas propuestas como nuevas especies no encajaron completamente con los de las especies descritas en este género. Además, el análisis de los genes estudiados respaldó la validez de las nuevas especies. El análisis multilocus de secuencias de los tres loci estudiados reforzó con mayor firmeza la definición de las nuevas especies. Conclusiones. Todos estos estudios confirman la separación de estas dos nuevas especies del resto de las especies descritas del género Malassezia, así como la existencia de especies dependientes de lípidos del género Malassezia en loros (AU)

Efficacy of dental unit waterlines disinfectants on a polymicrobial biofilm.

Due to their high surface-volume ratio, their laminar flow and frequent stagnation periods, dental unit waterlines (DUWL) foster the attachment of microorganisms and the development of biofilm, resulting in the continuous contamination of the outlet water from dental units; this contamination may be responsible for a potential risk of infection due to the exposure of patients and medical staff to droplet inhalation or splashed water. In this study, the anti-biofilm activity of three disinfectants recommended by dental unit manufacturers -Calbenium(©), Oxygenal 6(©) and Sterispray(©) - was evaluated. A dynamic model simulating DUWL conditions was developed and polymicrobial biofilms containing bacteria (Pseudomonas aeruginosa), fungi (Candida albicans) and Free Living Amoeba (FLA: Vermamoeba vermiformis) were allowed to form. The ability of disinfectants to reduce biofilm formation or to eradicate an already formed biofilm was evaluated. Results showed the various effects of the tested disinfectants according to their composition, concentration and the targeted species. V. vermiformis was resistant to disinfectants, regardless of the tested concentrations and the concentrations recommended by manufacturers were not the most appropriate. Results also showed that Calbenium(©) was the most effective disinfectant to reduce already formed biofilms; its maximum efficiency was observed from 0.5% on both P. aeruginosa and C. albicans compared to 2 and 3% respectively for Sterispray(©). The maximum efficiency of Oxygenal(©) was observed from 3% on P. aeruginosa but Oxygenal(©) was unable to totally eliminate C. albicans in the tested conditions, contrary to other disinfectants. Calbenium(©) was able to prevent biofilm formation efficiently even if it displayed no prophylactic activity against V. vermiformis. Overall, the FLA survival may contribute to maintaining other species. Finally the tested disinfectants were partially active against sessile microorganisms and more suitable concentrations could be used to increase their efficacy. Their use in a prophylactic rather than curative way should be recommended.

Antibiotic-resistant heterotrophic plate count bacteria and amoeba-resistant bacteria in aquifers of the Mooi River, North West province, South Africa.

Groundwater in the Mooi River catchment is prone to mining, agricultural, municipal and septic tank pollution. In this study physico-chemical and microbiological parameters were determined using appropriate methods. Bacterial isolates were identified by 16S rRNA sequencing (heterotrophic plate count (HPC) bacteria and amoeba-resistant bacteria (ARB)) and multiplex polymerase chain reaction (Escherichia coli). Antibiotic resistance tests were also performed. Physico-chemical parameters were generally within target water quality ranges for drinking water. HPC bacteria ranged between 10(5) and 10(7) colony-forming units (cfu)/ml. E. coli were enumerated from Trimpark, School and Cemetery. The Blaauwbank borehole was negative for faecal streptococci. Pseudomonas spp. were most abundant in the bulk water. Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying patterns of antibiotic resistance were observed. Most HPC bacterial isolates were resistant to cephalothin and/or amoxicillin and a few were resistant to erythromycin and streptomycin. Pseudomonas spp. was also the most abundant ARB. Other ARBs included Alcaligenes faecalis, Ochrobactrum sp. and Achromobacter sp. ARBs were resistant to streptomycin, chloramphenicol, cephalothin, and/or amoxicillin compared to HPCs. The presence of E. coli and ARB in these groundwater sources indicates potential human health risks. These risks should be further investigated and quantified, and groundwater should be treated before use.

Caracterización fisiológica y molecular de aislamientos de Phytophthora infestans de la región andina central colombiana / Physiological and molecular characterization of Phytophthora infestans isolates from the Central Colombian Andean Region

Antecedentes. El tizón tardío, causado por Phytophthora infestans, es una enfermedad devastadora de la papa y el tomate a nivel mundial, y en Colombia también ataca otros cultivos como la uchuva y el tomate de árbol. El conocimiento de la población del patógeno es determinante para el diseño efectivo de estrategias de control. Objetivos. Determinar las características fisiológicas y moleculares de aislamientos colombianos de P. infestans. Métodos. El nivel de resistencia al mefenoxam y al cimoxamil fue evaluado en aislamientos de Cundinamarca y Boyacá. Se estimó su virulencia y se determinó la producción y viabilidad de oosporas en diferentes sustratos con cruces entre aislamientos A1 y el aislamiento colombiano A2. Además, se determinó la diversidad molecular en el gen de avirulencia Avr3a, el gen de la β-tubulina y otros dos genes de copia única con motivo RXLR. Resultados. Los aislamientos colombianos tuvieron la posibilidad de reproducirse sexualmente. Encontramos todos los niveles de sensibilidad al mefenoxam, con el 48% de los aislamientos resistentes. Se detectó una diversidad de razas y a nivel genético la población fue clonal. Conclusiones. Estos resultados ayudarán a optimizar el uso de fungicidas y reducir la resistencia como estrategias de control, además de contribuir al conocimiento de la diversidad de este patógeno(AU)

Background. Late blight, caused by Phytophthora infestans, is one of the most devastating diseases found in potato and tomato crops worldwide. In Colombia it also attacks other important crops: cape gooseberry and tree tomato. The knowledge of the pathogen population is determinant to effectively design control strategies. Aims. To determine the physiological and molecular characteristics of a set of Colombian P. infestans isolates. Methods. Strains isolated from Cundinamarca and Boyacá were examined for the level of resistance to mefenoxam and cymoxanil. Virulence was tested for all strains and crosses between A1 mating type, from different hosts, and the Colombian A2 mating type were tested for the production and viability of oospores in different substrates. Additionally, the molecular diversity of the avirulence gene Avr3a, the β-tubulin gene, and two single copy genes showing RxLR motif, was assessed. Results. We found all levels of mefenoxam sensitivity, with 48% of the strains resistant. A high diversity of races was detected and the population was genetically clonal. Colombian strains had the possibility of sexual reproduction. Conclusions. These results will help in optimizing the use of fungicides and deployment of resistance as control strategies and will contribute to broader studies on diversity of this pathogen(AU)

Identificação e validação funcional de novos alvos das PKCs em célula tronco embrionária / Identification and functional validation of new targets of PKC in embryonic stem cell

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas

Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC

From parasite encounter to infection: multiple-scale drivers of parasite richness in a wild social primate population.

Host parasite diversity plays a fundamental role in ecological and evolutionary processes, yet the factors that drive it are still poorly understood. A variety of processes, operating across a range of spatial scales, are likely to influence both the probability of parasite encounter and subsequent infection. Here, we explored eight possible determinants of parasite richness, comprising rainfall and temperature at the population level, ranging behavior and home range productivity at the group level, and age, sex, body condition, and social rank at the individual level. We used a unique dataset describing gastrointestinal parasites in a terrestrial subtropical vertebrate (chacma baboons, Papio ursinus), comprising 662 fecal samples from 86 individuals representing all age-sex classes across two groups over two dry seasons in a desert population. Three mixed models were used to identify the most important factor at each of the three spatial scales (population, group, individual); these were then standardized and combined in a single, global, mixed model. Individual age had the strongest influence on parasite richness, in a convex relationship. Parasite richness was also higher in females and animals in poor condition, albeit at a lower order of magnitude than age. Finally, with a further halving of effect size, parasite richness was positively correlated to day range and temperature. These findings indicate that a range of factors influence host parasite richness through both encounter and infection probabilities but that individual-level processes may be more important than those at the group or population level.

Principales marcadores moleculares utilizados para la identificación de Babesia bovis y Babesia bigemina / Principal molecular markers used to identify Babesia bovis and Babesia bigemina

Este artículo describe los principales marcadores moleculares utilizados para la identificación de B. bovis y B. bigemina reportados en la literatura científica. Para ello se diseñó una revisión sistemática a partir de la aplicación de la estrategia metodológica PICO modificada con el objetivo de definir las secuencias nucleotídicas detectadas en los diferentes sitios geográficos y su utilidad diagnóstica. Se realizó una búsqueda avanzada con los términos “Babesia bovis” y “DNA” y “Babesia bigemina” y “DNA” en las bases de datos ScienceDirect, SpringerLink y PubMed que después de ser filtradas permitieron obtener un resultado total de 68 artículos originales. Tanto los artículos incluidos como los excluidos fueron almacenados en tablas, en las cuales se presenta la justificación de su condición dentro del estudio. A los 68 artículos seleccionados se les aplicó una evaluación con criterios de inclusión y exclusión previamente definidos, de este modo, 21 artículos originales cumplieron con los criterios de inclusión y se incluyeron en el estudio. Se describe la utilidad de los marcadores moleculares referenciados en la literatura científica desde 1995 hasta el 2010: la subunidad pequeña RNAr, el gen citocromo b, gen msa-1 and msa-2c, el gen Bv, el factor de elongación alfa (EF-1α), el gen de la beta-Tubulina, SBP 1-2-3, y los RAP; su aplicación diagnóstica y su utilización en los diferentes sitios geográficos. Los marcadores moleculares utilizados para la detección de las babesias bovinas varían dependiendo de la región geográfica, grado de conservación genética y resultados de estudios previos que concluyen su utilidad diagnóstica.

Protozoarios en aguas superficiales y muestras fecales de individuos de poblaciones rurales del municipio Montes, estado Sucre, Venezuela / Protozoans in superficial waters and faecal samples of individuals of rural populations of the Montes municipality, Sucre state, Venezuela

En el estado Sucre, el Río Manzanares se ve amenazado por actividades domésticas, agrícolas e industriales originadas por el hombre, convirtiéndose en factor de riesgo ambiental para sus habitantes. En este sentido se planteó evaluar la presencia de protozoarios en aguas superficiales de afluentes del Río Manzanares (Río Orinoco, Quebrada Seca, Río San Juan), municipio Montes, estado Sucre, Venezuela, así como también el análisis de muestras fecales de los habitantes de poblados aledaños. Se recolectaron muestras de aguas superficiales de los afluentes en estudio y fecales entre mayo 2006-abril 2007. Las muestras de aguas superficiales se procesaron con sedimentación por centrifugación, floculación y tinciones de Kinyoun y tricrómica; las muestras fecales se sometieron a examen directo con solución salina fisiológica y lugol, Ritchie modificado y las coloraciones antes mencionadas. Los protozoarios observados con mayor frecuencia en las aguas superficiales en los afluentes fueron: Amebas, Blastocystis sp., Endolimax sp., Chilomastix sp. y Giardia sp. Mientras que Blastocystis hominis, Endolimax nana y Entaomeba coli fueron los de mayor frecuencia observada en las muestras fecales. Los habitantes de Orinoco La Peña resultaron ser los más afectados por las infecciones parasitarias (77,60%), seguido de Río San Juan con 46,63%, y Quebrada Seca con 39,49%. La presencia de protozoarios patógenos y no patógenos en las aguas superficiales demuestra la contaminación fecal de los afluentes evaluados, por lo que representa un foco de infección permanente para los individuos que viven en las cercanías de estas aguas, esto se refleja por la observación de los mismos parásitos en ambas muestras.

In Sucre state, the Manzanares river is threatened by domestic, agricultural and industrial activities, becoming an environmental risk factor for its inhabitants. In this sense, the presence of protozoans in superficial waters of tributaries of the Manzanares river (Orinoco river, Quebrada Seca, San Juan river), Montes municipality, Sucre state, as well as the analysis of faecal samples from inhabitants of towns bordering these tributaries were evaluated. We collected faecal and water samples from may 2006 through april 2007. The superficial water samples were processed after centrifugation by the direct examination and floculation, using lugol, modified Kinyoun and trichromic colorations. Fecal samples where analyzed by direct examination with physiological saline solution and the modified Ritchie concentration method and using the other colorations techniques above mentioned. The most frequently observed protozoans in superficial waters in the three tributaries were: Amoebas, Blastocystis sp, Endolimax sp., Chilomastix sp. and Giardia sp. Whereas in faecal samples, Blastocystis hominis, Endolimax nana and Entaomeba coli had the greatest frequencies in the three communities. The inhabitants of Orinoco La Peña turned out to be most susceptible to these parasitic infections (77.60%), followed by San Juan River (46.63%) and Quebrada Seca (39.49%). The presence of pathogenic and nonpathogenic protozoans in superficial waters demonstrates the faecal contamination of the tributaries, representing a constant focus of infection for their inhabitants, inferred by the observation of the same species in both types of samples.

[Morphometric analysis of six natural populations of Difflugia tuberspinifera].

The morphometrical variability of six natural populations of Difflugia tuberspinifera was investigated using the statistic methods based on 374 samples from Yangtze River and Pearl River valleys. The size frequency distribution analysis indicated that D. tuberspinifera is a size-monomorphic species with normal distribution of shell height, shell diameter and aperture diameter. The size of spine length, collar height, foreside length and number of conical spines are the most variable. The correlation analysis showed that most characters are inter-correlated with P<0.05. The shell height, shell diameter, aperture diameter, spine length, collar height, rear end length, foreside length, number of aperture tooth-like structures and number of conical spines differed significantly between different populations (P<0.0001), but principal component analysis (PCA) did not clearly distinguish the six populations based on the morphometric data. However, the cluster analysis separated the six populations into two groups, the Pearl River-Yangtze River group and the Yangtze River group. Further, the populations with different shell characters can coexist within the same river valley. Thus, there is a high morphological variability or diversity within the populations of D. tuberspinifera which is related with local environmental conditions.

Strains of Uronema marinum (Scuticociliatia) co-isolated with amoebae of the genus Neoparamoeba.

Mixed infections of histophagous ciliates and Neoparamoeba spp. Page, 1987 were diagnosed in gill tissue of farmed turbot Psetta maxima (synonym: Scophthalmus maximus) and Atlantic salmon Salmo salar during a study of amoebic gill disease. Ciliates co-isolated from lesions grossly visible on gills of both fish hosts and from 2 species of red algae Lithophyllum racemus and Palmaria palmata were characterized morphologically and by using molecular markers. Sequences of small subunit (SSU) rDNA were determined for 6 strains of ciliates isolated from hosts collected in geographically distant localities. Of these, sequences of 5 strains revealed a surprisingly high level of similarity and identified the corresponding strains with Uronema marinum Lynn et Small, 1997. Thus, the set of environmental sequences of U. marinum available in the GenBank database to date was supplemented with the first sequences of potentially histophagous strains. On the basis of SSU rDNA, the 6th strain, also isolated from affected fish gills, was identified as Aristerostoma sp.

[Protozoans in superficial waters and faecal samples of individuals of rural populations of the Montes municipality, Sucre state, Venezuela]. / Protozoarios en aguas superficiales y muestras fecales de individuos de poblaciones rurales del municipio Montes, estado Sucre, Venezuela.

In Sucre state, the Manzanares river is threatened by domestic, agricultural and industrial activities, becoming an environmental risk factor for its inhabitants. In this sense, the presence of protozoans in superficial waters of tributaries of the Manzanares river (Orinoco river, Quebrada Seca, San Juan river), Montes municipality, Sucre state, as well as the analysis of faecal samples from inhabitants of towns bordering these tributaries were evaluated. We collected faecal and water samples from may 2006 through april 2007. The superficial water samples were processed after centrifugation by the direct examination and floculation, using lugol, modified Kinyoun and trichromic colorations. Fecal samples where analyzed by direct examination with physiological saline solution and the modified Ritchie concentration method and using the other colorations techniques above mentioned. The most frequently observed protozoans in superficial waters in the three tributaries were: Amoebas, Blastocystis sp, Endolimax sp., Chilomastix sp. and Giardia sp. Whereas in faecal samples, Blastocystis hominis, Endolimax nana and Entaomeba coli had the greatest frequencies in the three communities. The inhabitants of Orinoco La Peña turned out to be most susceptible to these parasitic infections (77.60%), followed by San Juan River (46.63%) and Quebrada Seca (39.49%). The presence of pathogenic and nonpathogenic protozoans in superficial waters demonstrates the faecal contamination of the tributaries, representing a constant focus of infection for their inhabitants, inferred by the observation of the same species in both types of samples.

Molecular phylogeny of asexual entomopathogenic fungi with special reference to Beauveria bassiana and Nomuraea rileyi

Basándose en el análisis de las secuencias de regiones parciales de los genes Beta-tubulina y ARN ribosómicosse estudió la filogenia, la taxonomía y las interrelaciones de importantes géneros de hongos entomopatógenosasexuales. Se estudio también la estructura de las especis Beauveria bassiana y Nomuraea rileyi. Seanalizó un total de 174 entradas fúngicas que representaban 94 especies. El análisis filogenético demostróque todas las especies de hongos entomopatogénicos asexuales incluidas en el estudio pertenecían a la familiaClavicipitaceae, del orden Hypocreales de los Ascomycota. Se observaron diferentes linajes dentro deB. bassiana, lo que demostró la complejidad de dicha especie. Algunos de los aislamientos de dicha especiedemostraron estar filogenéticamente más separados que determinados géneros morfológicos. Dentro de laespecie N. rileyi se pudo observar también la presencia de especiación críptica. Se concluye que los hongosasexuales entomopatogénicos han evolucionado de un linaje simple dentro de la familia Clavicipitaceae(AU)

The phylogenetic lineage, taxonomic affiliation and interrelationships of important asexualentomopathogenic fungal genera were studied using the sequences of partial regions of Beta-tubulin andrRNA genes. The species structures of Beauveria bassiana and Nomuraea rileyi were also investigated. Atotal of 147 fungal entries covering 94 species were analysed. Phylogenetic analysis placed all the asexualentomopathogenic fungal species analysed, in the family Clavicipitaceae of the order Hypocreales ofAscomycota. Deep phylogenetic lineages were observed in B. bassiana iterating the complex nature of thisspecies. Some of the isolates assigned to this species separated out more distinctly than morphologicallydistinguishable genera. Cryptic speciation was also evident in N. rileyi. It is concluded that the asexualfungi with entomopathogenic habit arose from a single lineage in sexual Clavicipitaceae(AU)