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.     

Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae)

An increase in clinical cases of Candidiosis globally as well as fungal resistance to drugs prompted the search for novel anti-Candida albicans agents from plant sources. Leaf extracts of Markhamia obtusifolia were screened for activity against C. albicans in vitro. An acetone extract obtained following serial exhaustive extraction contained mainly the active components with at least four active zones on the bioautogram. Bioassay guided fractionation of this extract led to the isolation of three compounds which inhibited the growth of three C. albicans strains. Based on spectroscopy studies (NMR and MS), the compounds were identified as 3β-hydroxyurs-12-en-28-oic acid, ursolic acid (1) 3β, 19α-dihydroxyurs-12-en-28-oic acid, pomolic acid (2) and 2β, 3β, 19α -trihydroxy-urs-12-en-28-oic acid, 2-epi-tormentic acid (3). The most active compound was 3β, 19α-dihydroxy-12-ursen-28-oic acid (2) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL for C. albicans isolated from dog and 25.0 µg/mL for C. albicans from cat and ATCC 90028 at 24 h following incubation. However, at 48 h of incubation MICs were > 400 µg/mL for all the three compounds isolated. This study indicated that M. obtusifolia could be a potential source of active principles against C. albicans.     

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.     

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.     

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.     

Real-time PCR detection of Holophagae (Acidobacteria) and Verrucomicrobia subdivision 1 groups in bulk and leek (Allium porrum) rhizosphere soils

In the light of the poor culturability of Acidobacteria and Verrucomicrobia species, group-specific real-time (qPCR) systems were developed based on the 16S rRNA gene sequences from culturable representatives of both groups. The number of DNA targets from three different groups, i.e. Holophagae (Acidobacteria group 8) and Luteolibacter/Prosthecobacter and unclassified Verrucomicrobiaceae subdivision 1, was determined in DNA extracts from different leek (Allium porrum) rhizosphere soil compartments and from bulk soil with the aim to determine the distribution of the three bacterial groups in the plant-soil ecosystem. The specificity of the designed primers was evaluated in three steps. First, in silico tests were performed which demonstrated that all designed primers 100% matched with database sequences of their respective groups, whereas lower matches with other non-target bacterial groups were found. Second, PCR amplification with the different primer sets was performed on genomic DNA extracts from target and from non-target bacteria. This test demonstrated specificity of the designed primers for the target groups, as single amplicons of expected sizes were found only for the target bacteria. Third, the qPCR systems were tested for specific amplifications from soil DNA extracts and 48 amplicons from each primer system were sequenced. All sequences were > 97% similar to database sequences of the respective target groups. Estimated cell numbers based on Holophagae-, Luteolibacter/Prosthecobacter- and unclassified Verrucomicrobiaceae subdivision 1-specific qPCRs from leek rhizosphere compartments and bulk soils demonstrated higher preference for one or both rhizosphere compartments above bulk soil for all three bacterial groups.     

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.     

RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Flavonoid profiles in the Heliohebe group of New Zealand Veronica (Plantaginaceae)

The Heliohebe group of Veronica (sect. Hebe) consists of five species occurring in the South Island of New Zealand. These species and a hybrid were analysed for their flavonoids. Five flavone glycosides were isolated and identified by NMR spectroscopy and three additional glycosides were detected by LC–UV–MS. Luteolin 7-O-, 3′-O- and 4′-O-glucosides and apigenin 7-O-glucoside were present in all six taxa investigated, 6-hydroxyluteolin glycosides were found in five and a luteolin caffeoylglycoside in four taxa, while a hypolaetin 7-O-glycoside was detected only in Veronica pentasepala. The 3′-O- and 4′-O-glucosides of luteolin are also common in other species of Veronica sect. Hebe (restricted to the Southern Hemisphere), but are rare in Northern Hemisphere species of Veronica and thus act as good chemotaxonomic markers for the section. The relatively simple flavonoid profiles found in the Heliohebe group are plesiomorphic and consistent with the group's status as sister to the Hebe clade. Based on the detected flavonoids, two groups could be distinguished within the Heliohebe clade: (1) Veronica hulkeana, Veronica lavaudiana and Veronica raoulii, characterised by luteolin caffeoylglycoside, and (2) V. pentasepala and Veronica scrupea, where this compound is replaced by a 6-hydroxyluteolin dihexoside.     

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5′ terminus of gce.In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

Synthesis and characterization of platinum(IV) complexes with N,S and S,S heterocyclic ligands

The reactions of [PtMe₃(OAc)(bpy)] (4) with the N,S and S,S containing heterocycles, pyrimidine-2-thione (pymtH), pyridine-2-thione (pytH), thiazoline-2-thione (tztH) and thiophene-2-thiol (tptH), resulted in the formation of the monomeric complexes [PtMe₃(-κS)(bpy)] ( = pymt, 5; pyt, 6; tzt, 7; tpt, 8), where the heterocyclic ligand is coordinated via the exocyclic sulfur atom. In contrast, in the reactions of [PtMe₃(OAc)(Me₂CO)_x] (3, x = 1 or 2) with pymtH, pytH, tztH and tptH dimeric complexes [{PtMe₃(μ-)}₂] (μ- = pymt, 9; pyt, 10; tzt, 11) and the tetrameric complex [{PtMe₃(μ₃-tpt-κS)}₄] (12), respectively, were formed. The complexes were characterized by microanalyses, ¹H and ¹³C NMR spectroscopy and negative ESI-MS (12) measurements. Single-crystal X-ray diffraction analysis of [PtMe₃(pymt-κS)(bpy)] (5) exhibited a conformation where the pymt ligand lies nearly perpendicular to the complex plane above the bpy ligand that was also confirmed by quantum chemical calculations on the DFT level of theory.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales.It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.