A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Naegleria fowleri: Light and electron microscopy study of mitosis

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.     

Free-living amoebae isolated from water-hyacinth root (Eichhornia crassipes)

Free-living amoebae are widely distributed in aquatic environments and their hygienic, medical and ecological relationships to man are increasingly important. The purpose of this study was to isolate free-living amoebae from water-hyacinth root (Eichhornia crassipes) and the water of an urban lake in Mexico City. Five grams of wet root were seeded on non-nutritive agar with Enterobacter aerogenes (NNE). Water samples were concentrated by centrifugation at 1200g for 15 min and the pellet was seeded on NNE. Of the 16 isolated genera, 10 were detected in both habitats. The most frequent were Vannella in root and Acanthamoeba and Naegleria in water. The total number of isolates and genera isolated from root was higher than that isolated from water. The differences between root and water are probably due to the morphological characteristics of water-hyacinth root, which provides a large habitat and refuge area for many organisms.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

First report of Gogia (Eocrinoidea, Echinodermata) from the Early-Middle Cambrian of Sonora (Mexico), with biostratigraphical and palaeoecological comments

The blastozoan echinoderm genus Gogia is reported for the first time in the Early and the Middle Cambrian of Mexico. Reports in different members of the section of San José de Gracia (Sonora State, northwestern Mexico) extend the palaeogeographical range of the genus to the South Laurentia, and the stratigraphic range of Gogia granulosa to the whole first half of middle Middle Cambrian. Isolated plates occur in rocks deposited in detrital inner platform and complete specimens, in carbonate outer platform, confirming their ability to live in diverse environments. Their presence in these different environments through the Early-Middle Cambrian on Laurentia agrees with the onshore-offshore expansion of echinoderms during Cambrian.     

中国柳莺属鸟类分类研究进展

柳莺属(Phylloscopus)是广泛分布于旧大陆地区的小型食虫鸟类,目前全世界已知66种,其中约50种见于亚洲。该属鸟类外部形态十分相近且同域分布种数较多,历来是鸟类分类学中的研究难点及热点。2003年贾陈喜等介绍了20世纪90年代以来发表的分布于我国的柳莺属鸟类新种及相关分类学变动,涉及3个新描述种和9个由亚种提升的种,共计30种26亚种。2003年以来关于柳莺属鸟类的分类学研究取得了许多新的成果,同时我国境内也发现了一些新的分布记录。综合最新的文献及分布资料,整理得知我国现有柳莺属鸟类41种31亚种,与2003年的状况相比已有较大的变化。主要表现在:新描述种方面,德国学者Martens等2008年描述了黄腹柳莺(P.affinis)种组中的一新种———华西柳莺(P.occisinensis);2010年瑞典学者Alstrm等报道在越南和老挝地区新发现的灰岩柳莺(P.calciatilis)已被证实在我国有分布;亚种提升为种方面,如日本柳莺(P.xanthodryas)自极北柳莺(P.borealis)中独立,冠纹柳莺(P.claudiae)和西南冠纹柳莺(P.reguloides)分开等;分类地位再评议方面,如灰头鹟莺(Seicercusxanthoschistos)实为灰头柳莺(P.xanthoschistos)等;新分布纪录方面,如欧柳莺(P.trochilus)在内蒙古达里诺尔湖地区发现确切记录等。现今对柳莺属鸟类分类学方面的研究已明显呈现出传统形态学、分子遗传学、声谱分析及野外鸣声回放实验等新老技术手段相整合,多方面多角度地阐述问题的局面。中国是世界柳莺属鸟种分布最多的国家(其中有9种仅在或主要于我国境内繁殖),但我国鸟类学者关于该属鸟类的研究工作并不多见。柳莺属鸟类在分类学和进化生物学等领域具有重要的研究价值,我国鸟类学工作者应当引起足够的重视和关注。     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

Structure and development of the style base in Abildgaardia, Bulbostylis, and Fimbristylis (Cyperaceae,Cyperoideae, Abildgaardieae)

The Abildgaardieae tribe within the family Cyperaceae comprises six or seven genera, among which Abildgaardia, Bulbostylis and Fimbristylis pose a challenge regarding their morphological delimitation. Molecular phylogenetic analyses including species of Abildgaardieae are rare, but in most of those studies, Abildgaardia and Fimbristylis appear as more closely related to each other than to the Bulbostylis genus. Duration of the style base has been one of the most widely used characters for delimiting these three genera. The style base is a persistent structure in most species of Bulbostylis and deciduous in Abildgaardia and Fimbristylis. The reasons why the style base may persist or fall off have been scarcely discussed. The assumption that abscission layers are present in the style base of all three genera and the fact that tracheids have been observed in the style base of Bulbostylis suggest that this structure might have histological complexity. In view of this, a complete ontogenetic and anatomical study of the gynoecium has been carried out for all these three genera. It turned out that the style base is histologically simple in Abildgaardia, Bulbostylis and Fimbristylis and shows similar structure and development in all three genera. The fact that the style base has a shorter duration in Abildgaardia and Fimbristylis than in Bulbostylis might be related to the lower number of sclerotised cells that make up such structures in the mature fruit of the former two genera. Abscission of the style and style base may be the result of much simpler reasons than the differentiation of an abscission layer, resulting merely from mechanical shear force effects. Differences among genera have been observed in the shape of the style base and the development of the style. The histological simplicity of the style base is consistent with the homoplastic appearance of this structure in genera that are not closely related (e.g. Rhynchospora). Because of this, while the presence of the thickened style base seems to be a synapomorphy in species of Abildgaardieae, its persistence on or detachment from the fruit might have emerged repeatedly during this clade evolution and might not be a suitable character for genera delimitation.     

Hydrozoan and protozoan epibionts on two decapod species, Liocarcinus depurator (Linnaeus, 1758) and Pilumnus hirtellus (Linnaeus, 1761), from Scotland

Crabs of the species Liocarcinus depurator and Pilumnus hirtellus, collected on the west coast of Scotland, showed hydrozoan and protozoan epibionts. The epibionts of Liocarcinus depurator were the protozoan Ephelota plana and the hydrozoans Clytia gracilis and Leuckartiara sp. The epibionts of Pilumnus hirtellus were the protozoans E. plana and Zoothamnium sp. For Liocarcinus depurator the number of Ephelota per crab fluctuated between 0 and 47 and the greatest number of suctorians were located on the chelipeds, carapace and anterior pereiopods. The hydrozoans, for Liocarcinus depurator, showed densities of 0-20 (Clytia gracilis on the second pereiopod) and 0-507 individuals per crab (Leuckartiara sp., principally on chelipeds, carapace and the fourth right pereiopod). For Pilumnus hirtellus, Ephelota plana showed densities between 3 and 56 individuals per crab, the greatest number of suctorians being located on the same areas as on Liocarcinus depurator. There was a density of 10-69 individuals per crab of Zoothamnium sp. on Pilumnus hirtellus (located on the carapace). Ephelota plana has not been observed previously as an epibiont on crustacea, nor had Zoothamnium sp. as an epibiont on Pilumnus hirtellus. Both hydrozoans, Leukartiara and Clytia, have not been previously described as epibionts on Liocarcinus depurator. Data about the morphological characteristics and distribution of these epibionts are included.     

Flower removal increases rhizome mass in natural populations of Alstroemeria aurea (Alstroemeriaceae)

Plant architecture and phenotypic plasticity under natural conditions remain little known for many rhizomatous species. This study evaluates, in situ, the plastic responses of Alstroemeria aurea plants from three Patagonian populations to flower or flowering-shoot removal. The size and architecture of treated and untreated plants were assessed. Nutrient contents (N, P and K) were evaluated for rhizomes and roots developed in two successive years. Those plants that were deprived of their inflorescences developed, on average, a heavier rhizome than both control plants and plants from which flowering shoots had been removed. Neither of the two treatments applied altered the number of metamers or the branching pattern of the rhizomes. The contents of N, P and K were higher in rhizomes than in roots. In summer, nutrients were more concentrated in inflorescences and the new rhizome segment than in the rhizome segment developed in the previous year. The idea that fruiting failure in A. aurea promotes resource re-assignment from aerial shoots to rhizomes without altering the architecture of plants is supported. The development of the underground portion of aerial shoots in late summer-autumn allows A. aurea plants to take full advantage of short growth periods, but would impose a limit to plasticity.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

蟛蜞菊属和孪花菊属(菊科-向日葵族)的细胞学研究

研究了菊科向日葵族鳢肠亚族蟛蜞菊属(Sphagneticola O. Hoffm.)和孪花菊属(Wollastonia DC. ex Decne.)各2种植物的染色体数目和染色体形态。蟛蜞菊[S. calendulacea (L.) Pruski]的染色体数目为2n=50, 核型公式为2n=18m+30sm+2st,南美蟛蜞菊[S. trilobata (L.) Pruski]的染色体数目为2n=56, 核型公式为2n=24m+28sm+4st, 孪花菊[W. biflora (L.) DC.]的染色体数目为2n=30,核型公式为2n=24m+4sm+2st,山孪花菊[W. montana (Blume) DC.]的染色体数目为2n=74, 核型公式为2n=37m+31sm+6st。根据上述结果并结合以前的有关资料,推测蟛蜞菊属的染色体基数可能为x=14和x=25,而不应是x=15。该属的3个新世界热带种[S. brachycarpa (Baker) Pruski、S. gracilis (Richard) Pruski和南美蟛蜞菊]可能都基于x=14, 其中S. gracilis为二倍体(2n=2x=28), S. brachycarpa和南美蟛蜞菊为四倍体(2n=4x=56); 唯一的亚洲种(蟛蜞菊)可能是基于x=25的二倍体(2n=2x=50)。染色体资料不支持将山孪花菊(x=37)这一植物置于孪花菊属(x=15)中。     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.