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1.  The evolutionary history of holometabolous insects inferred from transcriptome-based phylogeny and comprehensive morphological data 
Background
Despite considerable progress in systematics, a comprehensive scenario of the evolution of phenotypic characters in the mega-diverse Holometabola based on a solid phylogenetic hypothesis was still missing. We addressed this issue by de novo sequencing transcriptome libraries of representatives of all orders of holometabolan insects (13 species in total) and by using a previously published extensive morphological dataset. We tested competing phylogenetic hypotheses by analyzing various specifically designed sets of amino acid sequence data, using maximum likelihood (ML) based tree inference and Four-cluster Likelihood Mapping (FcLM). By maximum parsimony-based mapping of the morphological data on the phylogenetic relationships we traced evolutionary transformations at the phenotypic level and reconstructed the groundplan of Holometabola and of selected subgroups.
Results
In our analysis of the amino acid sequence data of 1,343 single-copy orthologous genes, Hymenoptera are placed as sister group to all remaining holometabolan orders, i.e., to a clade Aparaglossata, comprising two monophyletic subunits Mecopterida (Amphiesmenoptera + Antliophora) and Neuropteroidea (Neuropterida + Coleopterida). The monophyly of Coleopterida (Coleoptera and Strepsiptera) remains ambiguous in the analyses of the transcriptome data, but appears likely based on the morphological data. Highly supported relationships within Neuropterida and Antliophora are Raphidioptera + (Neuroptera + monophyletic Megaloptera), and Diptera + (Siphonaptera + Mecoptera). ML tree inference and FcLM yielded largely congruent results. However, FcLM, which was applied here for the first time to large phylogenomic supermatrices, displayed additional signal in the datasets that was not identified in the ML trees.
Conclusions
Our phylogenetic results imply that an orthognathous larva belongs to the groundplan of Holometabola, with compound eyes and well-developed thoracic legs, externally feeding on plants or fungi. Ancestral larvae of Aparaglossata were prognathous, equipped with single larval eyes (stemmata), and possibly agile and predacious. Ancestral holometabolan adults likely resembled in their morphology the groundplan of adult neopteran insects. Within Aparaglossata, the adult’s flight apparatus and ovipositor underwent strong modifications. We show that the combination of well-resolved phylogenies obtained by phylogenomic analyses and well-documented extensive morphological datasets is an appropriate basis for reconstructing complex morphological transformations and for the inference of evolutionary histories.
doi:10.1186/1471-2148-14-52
PMCID: PMC4000048  PMID: 24646345
2.  Emergence of sandflies (Phlebotominae) in Austria, a Central European country 
Parasitology Research  2013;112(12):4231-4237.
The possible existence of autochthonous sandfly populations in Central Europe north of the Alps has long been excluded. However, in the past years, sandflies have been documented in Germany, Belgium, and recently, also in Austria, close to the Slovenian border. Moreover, autochthonous human Leishmania and Phlebovirus infections have been reported in Central Europe, particularly in Germany. From 2010 to 2012, sandfly trapping (740 trap nights) was performed at 53 different capture sites in Austria using battery-operated CDC miniature light traps. Sites were chosen on the basis of their climate profile in the federal states Styria, Burgenland, and Lower Austria. Sandfly specimens found were transferred to 70 % ethanol for conservation. Identification was based on morphological characters of the male genitalia and the female spermathecae, respectively. Altogether, 24 specimens, 22 females and 2 males, all identified as Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, were found at six different sampling sites in all three federal states investigated. The highest number of catches was made on a farm in Lower Austria. Altogether, the period of sandfly activity in Austria was shown to be much longer than presumed, the earliest capture was made on July 3rd and the latest on August 28th. Sandflies have been autochthonous in Austria in small foci probably for long, but in the course of global warming, further spreading may be expected. Although P. mascittii is only an assumed vector of Leishmania spp.—data on its experimental transmission capacity are still lacking—the wide distribution of sandflies in Austria, a country thought to be free of sandflies, further supports a potential emergence of sandflies in Central Europe. This is of medical relevance, not only with respect to the transmission of Leishmania spp. for which a reservoir is given in dogs, but also with respect to the phleboviruses.
doi:10.1007/s00436-013-3615-9
PMCID: PMC3898358  PMID: 24126902
3.  Unexpected Increase of Alveolar Echincoccosis, Austria, 2011 
Emerging Infectious Diseases  2013;19(3):475-477.
Austria is part of the classical area of central Europe to which alveolar echinococcosis (AE) is endemic. Annual incidences in Austria were 2.4 and 2.8 cases/100,000 population during 1991–2000 and 2001–2010, respectively. Hence, the registration of 13 new AE patients in 2011 was unexpected. Increasing fox populations and past AE underreporting might have caused this increase.
doi:10.3201/eid1903.120595
PMCID: PMC3647648  PMID: 23628138
Alveolar echinococcosis; incidence; Austria; helminth; parasites; Echinococcus multilocularis; tapeworm
4.  New Species in the Old World: Europe as a Frontier in Biodiversity Exploration, a Test Bed for 21st Century Taxonomy 
PLoS ONE  2012;7(5):e36881.
The number of described species on the planet is about 1.9 million, with ca. 17,000 new species described annually, mostly from the tropics. However, taxonomy is usually described as a science in crisis, lacking manpower and funding, a politically acknowledged problem known as the Taxonomic Impediment. Using data from the Fauna Europaea database and the Zoological Record, we show that contrary to general belief, developed and heavily-studied parts of the world are important reservoirs of unknown species. In Europe, new species of multicellular terrestrial and freshwater animals are being discovered and named at an unprecedented rate: since the 1950s, more than 770 new species are on average described each year from Europe, which add to the 125,000 terrestrial and freshwater multicellular species already known in this region. There is no sign of having reached a plateau that would allow for the assessment of the magnitude of European biodiversity. More remarkably, over 60% of these new species are described by non-professional taxonomists. Amateurs are recognized as an essential part of the workforce in ecology and astronomy, but the magnitude of non-professional taxonomist contributions to alpha-taxonomy has not been fully realized until now. Our results stress the importance of developing a system that better supports and guides this formidable workforce, as we seek to overcome the Taxonomic Impediment and speed up the process of describing the planetary biodiversity before it is too late.
doi:10.1371/journal.pone.0036881
PMCID: PMC3359328  PMID: 22649502
5.  In vitro activity of N-chlorotaurine (NCT) in combination with NH4Cl against Trichomonas vaginalis 
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is usually treated with metronidazole, however resistance is on the rise. In this study, N-chlorotaurine (NCT), a new endogenous mild active chlorine compound for topical use, killed T. vaginalis in vitro within 15 min of treatment at a concentration of 55 mM (1%), which is well tolerated by human tissue. The activity of NCT was further enhanced by addition of ammonium chloride (NH4Cl). A combination of 5.5 mM (0.1%) NCT plus 19 mM (0.1%) NH4Cl killed 100% of trichomonads within 5 min.
doi:10.1016/j.ijantimicag.2010.09.006
PMCID: PMC3030749  PMID: 21074373
Trichomonas vaginalis; Susceptible; N-Chlorotaurine; Oxidant; In vitro
6.  Successful Treatment of Disseminated Acanthamoeba sp. Infection with Miltefosine 
Emerging Infectious Diseases  2008;14(11):1743-1746.
We report on an HIV-negative but immunocompromised patient with disseminated acanthamoebiasis, granulomatous amoebic encephalitis, and underlying miliary tuberculosis and tuberculous meningitis. The patient responded favorably to treatment with miltefosine, an alkylphosphocholine. The patient remained well with no signs of infection 2 years after treatment cessation.
doi:10.3201/eid1411.070854
PMCID: PMC2630722  PMID: 18976559
Acanthamoeba; granulomatous Acanthamoeba encephalitis (GAE); tuberculosis; immunocompromised non–HIV-infected patient; miltefosine; dispatch
7.  Cytotoxic Activity of N-Chlorotaurine on Acanthamoeba spp.▿  
Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK), which mainly occurs in contact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE), and disseminating diseases in the immunocompromised host. AK therapy is complex and irritating for the eye, skin lesions are difficult to treat, and there is no effective treatment for GAE. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the anti-Acanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. It was shown that NCT has amoebicidal qualities, both in phosphate-buffered saline (PBS) and in amoebic culture medium. After 6 h of treatment with 10 mM NCT in PBS, the levels of trophozoites of all strains investigated already showed at least a 2-log reduction. When the trophozoites were treated with 20 mM NCT in culture medium, they showed a 2-log reduction after 24 h. The addition of NH4Cl to NCT led to a faster decrease in the numbers of living cells, if tests were carried out in PBS. A delay of excystation was observed when cysts were treated with 55 mM (1%) NCT in culture medium. A complete failure of excystment was the result of treatment with 1% NCT plus 1% NH4Cl in PBS. Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated by human tissues and might be useful as a topical drug for the treatment of Acanthamoeba infections. The addition of ammonium chloride can be considered to enhance the activity.
doi:10.1128/AAC.00715-07
PMCID: PMC2224745  PMID: 18039920
8.  Granulomatous Amoebic Encephalitis Caused by Acanthamoeba Amoebae of Genotype T2 in a Human Immunodeficiency Virus-Negative Patient▿  
Journal of Clinical Microbiology  2007;46(1):338-340.
Acanthamoeba amoebae of genotype T2 were identified as the causative agent of Acanthamoeba skin lesions and granulomatous amoebic encephalitis (GAE) in a human immunodeficiency virus-negative patient with underlying tuberculosis. To our knowledge this, is the first case of GAE involving genotype T2.
doi:10.1128/JCM.01177-07
PMCID: PMC2224275  PMID: 18003807
9.  Onchocerca jakutensis Filariasis in Humans 
Emerging Infectious Diseases  2007;13(11):1749-1752.
We identified Onchocerca jakutensis as the causative agent of an unusual human filariasis in a patient with lupus erythematosus. To our knowledge, this is the first case of human infection with O. jakutensis and the first human case of zoonotic onchocercosis involving >1 worm.
doi:10.3201/eid1311.070017
PMCID: PMC3375810  PMID: 18217562
Onchocerca jakutensis; zoonosis; filariasis; PCR; paraffin-embedded tissue; lupus erythematosus; dispatch
10.  Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe 
Emerging Infectious Diseases  2003;9(8):943-948.
In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.
doi:10.3201/eid0908.020748
PMCID: PMC3020600  PMID: 12967491
babesiosis; Babesia divergens; Babesia odocoilei; Babesia venatorum; EU1; Italy; Austria; taxonomy; Ixodes ricinus; 18S rRNA gene; research
11.  Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp. 
Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.
doi:10.1128/AAC.46.3.695-701.2002
PMCID: PMC127497  PMID: 11850250
12.  Correlations between Morphological, Molecular Biological, and Physiological Characteristics in Clinical and Nonclinical Isolates of Acanthamoeba spp. 
Applied and Environmental Microbiology  2000;66(10):4408-4413.
Eleven Acanthamoeba isolates, obtained from Acanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed Acanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.
PMCID: PMC92317  PMID: 11010891
13.  Blinded, Externally Controlled Multicenter Evaluation of Light Microscopy and PCR for Detection of Microsporidia in Stool Specimens 
Journal of Clinical Microbiology  1998;36(6):1814-1818.
The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.
PMCID: PMC104932  PMID: 9620432

Results 1-13 (13)