Jaroslav Kulda - Academia.edu (original) (raw)

Papers by Jaroslav Kulda

Research paper thumbnail of Employment of Experimental Animals in Studies of Trichomonas vaginalis Infection

Trichomonads Parasitic in Humans, 1990

Research paper thumbnail of Remarks on the diplomastigine flagellates from the intestine of fishes

Research paper thumbnail of Dientamoeba fragilis and Other Intestinal Flagellates

Topley & Wilson's Microbiology and Microbial Infections, 2010

Research paper thumbnail of ThALES—three axis low energy spectroscopy at the Institut Laue–Langevin

Measurement Science and Technology, 2008

Building on the strength of the present cold neutron three-axis spectrometer IN14, but using stat... more Building on the strength of the present cold neutron three-axis spectrometer IN14, but using state-of-the-art neutron optics, we conceived the next generation three-axis instrument for low energy spectroscopy (ThALES) at the Institut Laue-Langevin (ILL). The main aims of the new instrument are: (i) to increase the overall data collection rate by rebuilding the neutron optics of the primary spectrometer achieving a higher incident neutron flux as well as by multiplexing the analyser-detector system, (ii) to provide an efficient and easy-to-use polarized neutron option, (iii) to extend the incident neutron range towards higher energies bridging the gap with thermal instruments, and (iv) to be able to use high-field magnets---such as the currently available 15 T cryomagnet---under all possible experimental conditions, i.e. in a wider range of incident energies. The expected increase in count rate by at least one order of magnitude allows for new experiments such as high pressure experiments on small sample sizes or investigations of magnetic excitations in thin films. Polarized inelastic neutron measurements should equal count rates of the present IN14 in unpolarized mode. The implementation of various optical elements enhances the flexibility of the instrument and allows trading momentum resolution for high neutron intensity.

Research paper thumbnail of Monte carlo simulations of resolution functions and scan profiles for neutron three-axis spectrometers

Journal of Neutron Research, 1997

Research paper thumbnail of Virtual experiments: the ultimate aim of neutron ray-tracing simulations

Journal of Neutron Research, 2008

We define a virtual neutron experiment as a complete simulation of an experiment, from source ove... more We define a virtual neutron experiment as a complete simulation of an experiment, from source over sample to detector. The virtual experiment (VE) will ideally interface with the instrument control software for the input and with standard data analysis packages for the virtual data output. Virtual experiments are beginning to make their way into neutron scattering science with applications as diverse as instrument design/upgrade, experiment planning, data analysis, test of analysis software, teaching, and outreach. In this paper, we summarize the recent developments in this field and make suggestions for future developments and use of VEs.

Research paper thumbnail of The use of random amplified polymorphic DNA (RAPD) analysis for studies of genetic variation in populations of the blowfly Lucilia sericata (Diptera: Calliphoridae) in southern England

Bulletin of Entomological Research, 1995

The use of the random amplified polymorphic DNA (RAPD) polymerase chain reaction to characterize ... more The use of the random amplified polymorphic DNA (RAPD) polymerase chain reaction to characterize individual Lucilia sericata Meigen from southern England was evaluated. Some simple techniques which allowed the preservation and extraction of DNA to be optimized without the complications of transporting liquid nitrogen were investigated. The RAPD results show that closely related L. sericata, including those from a single strike, can be readily distinguished from each other on the basis of their RAPD profiles resolved using electrophoretic analysis; profiles were defined with ten random primers. Analysis of these RAPD data using a similarity coefficient method and a recently developed randomization test to detect the non-random association of alleles at different loci, allowed the genetic homogeneity of L. sericata within southern Britain to be explored. This study shows that while a number of factors can complicate the use and interpretation of RAPD fragments as genetic markers, RAPD fingerprinting can be a valuable technique for studies of intraspecific genetic variation in L. sericata.

Research paper thumbnail of Retortamonad Flagellates are Closely Related to Diplomonads— Implications for the History of Mitochondrial Function in Eukaryote Evolution

We present the first molecular phylogenetic examination of the evolutionary position of retortamo... more We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weights methods but was marginally rejected by parametric bootstrapping. Analyses with additional phylogenetic markers are needed to test this controversial branching order within the retortamonad ϩ diplomonad clade. Nevertheless, the robust phylogenetic association between diplomonads and retortamonads suggests that they share an amitochondriate ancestor. Because strong evidence indicates that diplomonads have secondarily lost their mitochondria (rather than being ancestrally amitochondriate), our results imply that retortamonads are also secondarily amitochondriate. Of the various groups of eukaryotes originally suggested to be primitively amitochondriate under the archezoa hypothesis, all have now been found to have physical or genetic mitochondrial relics (or both) or form a robust clade with an organism with such a relic.

Research paper thumbnail of Critical Taxonomic Revision of Parabasalids with Description of one New Genus and three New Species

We propose a new classification of Parabasalia which is congruent with both ultrastructural and m... more We propose a new classification of Parabasalia which is congruent with both ultrastructural and molecular-phylogenetic studies. We identify six main parabasalid lineages and give them the rank of class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichonymphea, and Spirotrichonymphea. Trichomonadea is characterized by a single mastigont and by the absence of both a comb-like structure and an infrakinetosomal body. Most representatives also possess a lamelliform undulating membrane. Trichomonadea is divided into two monophyletic orders, Trichomonadida (family Trichomonadidae; with a B-type costa) and Honigbergiellida (families Honigbergiellidae, Hexamastigidae and Tricercomitidae; without a costa). The class Tritrichomonadea, with a single order Tritrichomonadida, is ancestrally characterized by a single mastigont with four flagella, and both a comb-like structure and an infrakinetosomal body. The morphologically most complex representatives (family Tritrichomonadidae) possess in addition a rail-type undulating membrane, an A-type costa, and a suprakinetosomal body. These last three characters are absent in families Monocercomonadidae and Simplicimonadidae. The remaining tritrichomonadids, Dientamoebidae, have undergone reductive evolution. Cristamonads (Cristamonadea) are morphologically derived from tritrichomonads. Because we are unable to determine morphologically homogenous monophyletic lineages within cristamonads, we classify all cristamonads into a single family, Lophomonadidae. Hypotrichomonadea, comprising the genera Trichomitus and Hypotrichomonas, resembles Tritrichomonadea by an A-type costa, and by the presence of a comb-like structure in the mastigont. However, they do not possess an infrakinetosomal body, and are not specifically related to Tritrichomonadea in molecular-phylogenetic analyses. Moreover, unlike Tritrichomonadea, Hypotrichomonadea possesses a lamelliform undulating membrane. The remaining parabasalids are of complex morphology and belong to the classes Trichonymphea and Spirotrichonymphea. A new parabasalid genus, Simplicimonas (Tritrichomonadea), and three new species, Tetratrichomonas undula, Hexamastix coercens and Simplicimonas similis, are described.

Research paper thumbnail of Inference of the Phylogenetic Position of Oxymonads Based on Nine Genes: Support for Metamonada and Excavata

Molecular Biology and Evolution, 2005

Circumscribing major eukaryote groups and resolving higher order relationships between them are a... more Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most chal- lenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding

Research paper thumbnail of Characterization of Trichomonad Species and Strains by PCR Fingerprinting

Journal of Eukaryotic Microbiology, 2007

Research paper thumbnail of Morphological and Molecular Diversity of the Monocercomonadid Genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov

Protist, 2007

Family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa... more Family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane, both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of SSU rDNA and ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: 1) Monocercomonas colubrorum, 2) Monocercomonas ruminantium together with a strain ATCC 50321 designed as Pseudotrichomonas keilini and 3) Hexamastix. Twenty isolates of Monocercomonas colubrorum divided into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of Monocercomonadidae confirmed that the absence of costa and undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters -infrakinetosomal body, comblike structure, marginal lamella and the type of axostyle -are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters. (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52: 399-451. Brugerolle G (1973) Sur l'existence de vrais kystes chez les Trichomonadines intestinales. Ultrastructure des kystes de Trichomitus batrachorum Perty 1852, Trichomitus sanguisugae Alexeieff 1911, et Monocercomonas tipulae Mackinnon 1910. C R Acad Sci Paris 277: 2193-2196 Brugerolle G (1976) Cytologie ultrastructurale, systematique et evolution des Trichomonadida. Annales de la Station Biologique Besse-en-Chandesse 10: 1-57 Brugerolle G (1991) Cell organization in free-living amitochondriate heterotrophic flagellates. In Patterson D and Larsen J (eds) The Biology of Free-living Heterotrophic Flagellates. Clarendon Press, Oxford, pp 133-148 Brugerolle G and Patterson PJ (2001) Ultrastructure of Joenina pulchella Grassi, 1917 (Protista, Parabasalia), a reassessment of evolutionary trends in the parabasalids, and a new order Cristamonadida for devescovinid, calonymphid and lophomonad flagellates. Org Divers Evol 1:147-160 Clark CG and Diamond LS (2002) Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 15: 329-341 Dacks JB and Redfield RJ (1998) Phylogenetic placement of Trichonympha. J Eukaryot Microbiol 45: 445-447 Delgado-Viscogliosi P, Viscogliosi E, Gerbod D, Kulda J, Sogin ML, Edgcomb VP (2000) Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily. J Eukaryot Microbiol 47: 70-75 Dolan M, Wier AM, Melnitsky H, Whiteside JH, Margulis L (2004) Cysts and symbionts of Staurojoenina assimilis Kirby from Neotermes. Eur J Protistol 40: 257-264 Edgcomb VP, Viscogliosi E, Simpson AGB, Delgado-Viscogliosi P, Roger AJ, Sogin ML (1998) New insights into the phylogeny of trichomonads inferred from small subunit rRNA sequences. Protist 149:359-366 Farmer MA (1993). Ultrastructure of Ditrichomonas honigbergii n. g., n. sp. (Parabasalia) and its relationship to amitochondrial protists. J Eukaryot Microbiol 40: 619-626 Felleisen RSJ (1997) Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitology 115:111-119 Felsenstein J (1978) Cases in which parsimony or compatibility methods will be positively misleading. Syst Zool 27:401-410 Gerbod D, Edgcomb VP, Noël C, Delgado-Viscogliosi P, Viscogliosi E (2000) Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences. Int Microbiol 3:165-172 Gerbod D, Edgcomb VP, Noël C, Zenner L, Wintjens R, Delgado-Viscogliosi P, Holder ME, Sogin ML, Viscogliosi E (2001) Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence. J Euk Microbiol 48:498-504 Gerbod D, Noël C, Dolan MF, Edgcomb VP, Kitade O, Noda S, Dufernez F, Ohkuma M, Kudo T, Capron M, Sogin ML, Viscogliosi E (2002) Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea). Mol Phylogenet Evol 25:545-556 Gerbod D, Sanders E, Moriya S, Noël C, Takasu H, Fast NM, Delargo-Viscogliosi P, Ohkuma M, Kudo T, Capron M, Palmer JD, Keeling PJ, Viscogliosi E (2004) Molecular phylogenies of Parabasalia inferred from four protein genes and comparison with rRNA trees. Mol Phylogenet Evol 31:572-580 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95-98 Hampl V, Cepicka I, Flegr J, Tachezy J, Kulda J (2004) Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree. Mol Phylogenet Evol 32:711-723 Hampl V, Vrlík M, Cepicka I, Pecka Z, Kulda J, Tachezy J (2006) Affiliation of Cochlosoma to trichomonads confirmed by phylogenetic analysis of the small subunit rRNA gene and a new family concept of the order Trichomonadida. Int J Syst Evol Microbiol 56: 305-312. Hollande A and Caruette-Valentin J (1971) Les atractophores, l'induction du fuseau, et la division cellulaire chez les Hypermastigines, étude infrastructurale et révision systématique des Trichonymphines et des Spirotrichonymphines. Protistologica 7:5-100 Honigberg BM (1955) Structure and morphogenesis of two new species of Hexamastix from lizards. J Parasitol 41: 1-17 Honigberg BM (1963) Evolutionary and Systematic Relationships in the Flagellate Order Trichomonadida Kirby. J Protozool 10: 20-63 Honigberg BM, Christian HH (1954) Characteristics of Hexamastix batrachorum (Alexeieff). J Parasitol 40: 508-514 Jensen EA, Hammond DM (1964) A morphological study of trichomonads and related flagellates from the bovine digestive tract. J Protozool 11: 386-394 Keeling PJ (2002) Molecular phylogenetic position of Trichomitopsis termopsidis (Parabasalia) and evidence for the Trichomitopsiinae. Eur J Protistol 38: 279-286

Research paper thumbnail of New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas

Molecular Phylogenetics and Evolution, 2006

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from... more We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mammalian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and α-tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus Trichomonas.

Research paper thumbnail of Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins

The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate... more The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H2 ,C O 2 and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic

Research paper thumbnail of Retortamonad flagellates are closely related to diplomonads--implications for the history of mitochondrial function in eukaryote evolution

Molecular biology and evolution, 2002

We present the first molecular phylogenetic examination of the evolutionary position of retortamo... more We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weight...

Research paper thumbnail of New evolutionary lineages, unexpected diversity, and host speciWcity in the parabasalid genus Tetratrichomonas

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from... more We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mamma- lian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and -tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus

Research paper thumbnail of Cryptic species within the Tetratrichomonas gallinarum species complex revealed by molecular polymorphism

Fuel and Energy Abstracts, 2005

Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds... more Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds. The pathogenicity of this species is controversial, presenting an unsettled problem as yet. We analysed the polymorphism and genetic relationship among 29 isolates of T. gallinarum obtained from eight bird species and five T. gallinarum-like isolates from the oral cavity and lower respiratory tract of human patients. Two methods were used for the analyses: RAPD and sequencing of 16S rRNA, 5.8S rRNA, ITS1 and ITS2 genes, both producing consistent and well-supported results. The isolates were divided into five groups, A–E, with eleven subgroups. The distance between groups E, D and the cluster A–B–C considerably exceeded usual intraspecific polymorphism seen in trichomonads. Moreover, the largest subgroup, A2 (containing 18 isolates), was divided into three branches according to the host specificity. All isolates from humans were placed into avian subgroups A2 and B2. We conclude that ou...

Research paper thumbnail of Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization

Veterinary Parasitology, 2010

Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary ... more Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.

Research paper thumbnail of Molecular Phytogeny of Parabasalids Based on Small Subunit rRNA Sequences, with Emphasis on the Trichomonadinae Subfamily

The Journal of Eukaryotic Microbiology, 2000

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus h... more We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus hatrachorum strain R 105, Trtratrichonzonas gallinarum, and Pentatrichomonas horninis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The-Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trickomonas species as supported by morphological data, with P. horninis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. hatrackorum within the Trichomonadinae. Trickomitus butrackorum (strains R10S and BUB) and Hypotrichornonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. hatrachorum precedes the Trichomonadinae-Tritrichornonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympku, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrickomonus ,fiptu.s. Monocercomonas sp., Dientamoeha ,fragilis, and the unidentified Reticulitermes javipes gut symbiont 1 remain unclear.

Research paper thumbnail of Genetic Analysis of Giardia from Hoofed Farm Animals Reveals Artiodactyl-Specific and Potentially Zoonotic Genotypes

Journal of Eukaryotic Microbiology, 2007

Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture o... more Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A-one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from humanderived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group I1 G. inresrinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.

Research paper thumbnail of Employment of Experimental Animals in Studies of Trichomonas vaginalis Infection

Trichomonads Parasitic in Humans, 1990

Research paper thumbnail of Remarks on the diplomastigine flagellates from the intestine of fishes

Research paper thumbnail of Dientamoeba fragilis and Other Intestinal Flagellates

Topley & Wilson's Microbiology and Microbial Infections, 2010

Research paper thumbnail of ThALES—three axis low energy spectroscopy at the Institut Laue–Langevin

Measurement Science and Technology, 2008

Building on the strength of the present cold neutron three-axis spectrometer IN14, but using stat... more Building on the strength of the present cold neutron three-axis spectrometer IN14, but using state-of-the-art neutron optics, we conceived the next generation three-axis instrument for low energy spectroscopy (ThALES) at the Institut Laue-Langevin (ILL). The main aims of the new instrument are: (i) to increase the overall data collection rate by rebuilding the neutron optics of the primary spectrometer achieving a higher incident neutron flux as well as by multiplexing the analyser-detector system, (ii) to provide an efficient and easy-to-use polarized neutron option, (iii) to extend the incident neutron range towards higher energies bridging the gap with thermal instruments, and (iv) to be able to use high-field magnets---such as the currently available 15 T cryomagnet---under all possible experimental conditions, i.e. in a wider range of incident energies. The expected increase in count rate by at least one order of magnitude allows for new experiments such as high pressure experiments on small sample sizes or investigations of magnetic excitations in thin films. Polarized inelastic neutron measurements should equal count rates of the present IN14 in unpolarized mode. The implementation of various optical elements enhances the flexibility of the instrument and allows trading momentum resolution for high neutron intensity.

Research paper thumbnail of Monte carlo simulations of resolution functions and scan profiles for neutron three-axis spectrometers

Journal of Neutron Research, 1997

Research paper thumbnail of Virtual experiments: the ultimate aim of neutron ray-tracing simulations

Journal of Neutron Research, 2008

We define a virtual neutron experiment as a complete simulation of an experiment, from source ove... more We define a virtual neutron experiment as a complete simulation of an experiment, from source over sample to detector. The virtual experiment (VE) will ideally interface with the instrument control software for the input and with standard data analysis packages for the virtual data output. Virtual experiments are beginning to make their way into neutron scattering science with applications as diverse as instrument design/upgrade, experiment planning, data analysis, test of analysis software, teaching, and outreach. In this paper, we summarize the recent developments in this field and make suggestions for future developments and use of VEs.

Research paper thumbnail of The use of random amplified polymorphic DNA (RAPD) analysis for studies of genetic variation in populations of the blowfly Lucilia sericata (Diptera: Calliphoridae) in southern England

Bulletin of Entomological Research, 1995

The use of the random amplified polymorphic DNA (RAPD) polymerase chain reaction to characterize ... more The use of the random amplified polymorphic DNA (RAPD) polymerase chain reaction to characterize individual Lucilia sericata Meigen from southern England was evaluated. Some simple techniques which allowed the preservation and extraction of DNA to be optimized without the complications of transporting liquid nitrogen were investigated. The RAPD results show that closely related L. sericata, including those from a single strike, can be readily distinguished from each other on the basis of their RAPD profiles resolved using electrophoretic analysis; profiles were defined with ten random primers. Analysis of these RAPD data using a similarity coefficient method and a recently developed randomization test to detect the non-random association of alleles at different loci, allowed the genetic homogeneity of L. sericata within southern Britain to be explored. This study shows that while a number of factors can complicate the use and interpretation of RAPD fragments as genetic markers, RAPD fingerprinting can be a valuable technique for studies of intraspecific genetic variation in L. sericata.

Research paper thumbnail of Retortamonad Flagellates are Closely Related to Diplomonads— Implications for the History of Mitochondrial Function in Eukaryote Evolution

We present the first molecular phylogenetic examination of the evolutionary position of retortamo... more We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weights methods but was marginally rejected by parametric bootstrapping. Analyses with additional phylogenetic markers are needed to test this controversial branching order within the retortamonad ϩ diplomonad clade. Nevertheless, the robust phylogenetic association between diplomonads and retortamonads suggests that they share an amitochondriate ancestor. Because strong evidence indicates that diplomonads have secondarily lost their mitochondria (rather than being ancestrally amitochondriate), our results imply that retortamonads are also secondarily amitochondriate. Of the various groups of eukaryotes originally suggested to be primitively amitochondriate under the archezoa hypothesis, all have now been found to have physical or genetic mitochondrial relics (or both) or form a robust clade with an organism with such a relic.

Research paper thumbnail of Critical Taxonomic Revision of Parabasalids with Description of one New Genus and three New Species

We propose a new classification of Parabasalia which is congruent with both ultrastructural and m... more We propose a new classification of Parabasalia which is congruent with both ultrastructural and molecular-phylogenetic studies. We identify six main parabasalid lineages and give them the rank of class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichonymphea, and Spirotrichonymphea. Trichomonadea is characterized by a single mastigont and by the absence of both a comb-like structure and an infrakinetosomal body. Most representatives also possess a lamelliform undulating membrane. Trichomonadea is divided into two monophyletic orders, Trichomonadida (family Trichomonadidae; with a B-type costa) and Honigbergiellida (families Honigbergiellidae, Hexamastigidae and Tricercomitidae; without a costa). The class Tritrichomonadea, with a single order Tritrichomonadida, is ancestrally characterized by a single mastigont with four flagella, and both a comb-like structure and an infrakinetosomal body. The morphologically most complex representatives (family Tritrichomonadidae) possess in addition a rail-type undulating membrane, an A-type costa, and a suprakinetosomal body. These last three characters are absent in families Monocercomonadidae and Simplicimonadidae. The remaining tritrichomonadids, Dientamoebidae, have undergone reductive evolution. Cristamonads (Cristamonadea) are morphologically derived from tritrichomonads. Because we are unable to determine morphologically homogenous monophyletic lineages within cristamonads, we classify all cristamonads into a single family, Lophomonadidae. Hypotrichomonadea, comprising the genera Trichomitus and Hypotrichomonas, resembles Tritrichomonadea by an A-type costa, and by the presence of a comb-like structure in the mastigont. However, they do not possess an infrakinetosomal body, and are not specifically related to Tritrichomonadea in molecular-phylogenetic analyses. Moreover, unlike Tritrichomonadea, Hypotrichomonadea possesses a lamelliform undulating membrane. The remaining parabasalids are of complex morphology and belong to the classes Trichonymphea and Spirotrichonymphea. A new parabasalid genus, Simplicimonas (Tritrichomonadea), and three new species, Tetratrichomonas undula, Hexamastix coercens and Simplicimonas similis, are described.

Research paper thumbnail of Inference of the Phylogenetic Position of Oxymonads Based on Nine Genes: Support for Metamonada and Excavata

Molecular Biology and Evolution, 2005

Circumscribing major eukaryote groups and resolving higher order relationships between them are a... more Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most chal- lenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding

Research paper thumbnail of Characterization of Trichomonad Species and Strains by PCR Fingerprinting

Journal of Eukaryotic Microbiology, 2007

Research paper thumbnail of Morphological and Molecular Diversity of the Monocercomonadid Genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov

Protist, 2007

Family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa... more Family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane, both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of SSU rDNA and ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: 1) Monocercomonas colubrorum, 2) Monocercomonas ruminantium together with a strain ATCC 50321 designed as Pseudotrichomonas keilini and 3) Hexamastix. Twenty isolates of Monocercomonas colubrorum divided into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of Monocercomonadidae confirmed that the absence of costa and undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters -infrakinetosomal body, comblike structure, marginal lamella and the type of axostyle -are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters. (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52: 399-451. Brugerolle G (1973) Sur l'existence de vrais kystes chez les Trichomonadines intestinales. Ultrastructure des kystes de Trichomitus batrachorum Perty 1852, Trichomitus sanguisugae Alexeieff 1911, et Monocercomonas tipulae Mackinnon 1910. C R Acad Sci Paris 277: 2193-2196 Brugerolle G (1976) Cytologie ultrastructurale, systematique et evolution des Trichomonadida. Annales de la Station Biologique Besse-en-Chandesse 10: 1-57 Brugerolle G (1991) Cell organization in free-living amitochondriate heterotrophic flagellates. In Patterson D and Larsen J (eds) The Biology of Free-living Heterotrophic Flagellates. Clarendon Press, Oxford, pp 133-148 Brugerolle G and Patterson PJ (2001) Ultrastructure of Joenina pulchella Grassi, 1917 (Protista, Parabasalia), a reassessment of evolutionary trends in the parabasalids, and a new order Cristamonadida for devescovinid, calonymphid and lophomonad flagellates. Org Divers Evol 1:147-160 Clark CG and Diamond LS (2002) Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 15: 329-341 Dacks JB and Redfield RJ (1998) Phylogenetic placement of Trichonympha. J Eukaryot Microbiol 45: 445-447 Delgado-Viscogliosi P, Viscogliosi E, Gerbod D, Kulda J, Sogin ML, Edgcomb VP (2000) Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily. J Eukaryot Microbiol 47: 70-75 Dolan M, Wier AM, Melnitsky H, Whiteside JH, Margulis L (2004) Cysts and symbionts of Staurojoenina assimilis Kirby from Neotermes. Eur J Protistol 40: 257-264 Edgcomb VP, Viscogliosi E, Simpson AGB, Delgado-Viscogliosi P, Roger AJ, Sogin ML (1998) New insights into the phylogeny of trichomonads inferred from small subunit rRNA sequences. Protist 149:359-366 Farmer MA (1993). Ultrastructure of Ditrichomonas honigbergii n. g., n. sp. (Parabasalia) and its relationship to amitochondrial protists. J Eukaryot Microbiol 40: 619-626 Felleisen RSJ (1997) Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitology 115:111-119 Felsenstein J (1978) Cases in which parsimony or compatibility methods will be positively misleading. Syst Zool 27:401-410 Gerbod D, Edgcomb VP, Noël C, Delgado-Viscogliosi P, Viscogliosi E (2000) Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences. Int Microbiol 3:165-172 Gerbod D, Edgcomb VP, Noël C, Zenner L, Wintjens R, Delgado-Viscogliosi P, Holder ME, Sogin ML, Viscogliosi E (2001) Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence. J Euk Microbiol 48:498-504 Gerbod D, Noël C, Dolan MF, Edgcomb VP, Kitade O, Noda S, Dufernez F, Ohkuma M, Kudo T, Capron M, Sogin ML, Viscogliosi E (2002) Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea). Mol Phylogenet Evol 25:545-556 Gerbod D, Sanders E, Moriya S, Noël C, Takasu H, Fast NM, Delargo-Viscogliosi P, Ohkuma M, Kudo T, Capron M, Palmer JD, Keeling PJ, Viscogliosi E (2004) Molecular phylogenies of Parabasalia inferred from four protein genes and comparison with rRNA trees. Mol Phylogenet Evol 31:572-580 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95-98 Hampl V, Cepicka I, Flegr J, Tachezy J, Kulda J (2004) Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree. Mol Phylogenet Evol 32:711-723 Hampl V, Vrlík M, Cepicka I, Pecka Z, Kulda J, Tachezy J (2006) Affiliation of Cochlosoma to trichomonads confirmed by phylogenetic analysis of the small subunit rRNA gene and a new family concept of the order Trichomonadida. Int J Syst Evol Microbiol 56: 305-312. Hollande A and Caruette-Valentin J (1971) Les atractophores, l'induction du fuseau, et la division cellulaire chez les Hypermastigines, étude infrastructurale et révision systématique des Trichonymphines et des Spirotrichonymphines. Protistologica 7:5-100 Honigberg BM (1955) Structure and morphogenesis of two new species of Hexamastix from lizards. J Parasitol 41: 1-17 Honigberg BM (1963) Evolutionary and Systematic Relationships in the Flagellate Order Trichomonadida Kirby. J Protozool 10: 20-63 Honigberg BM, Christian HH (1954) Characteristics of Hexamastix batrachorum (Alexeieff). J Parasitol 40: 508-514 Jensen EA, Hammond DM (1964) A morphological study of trichomonads and related flagellates from the bovine digestive tract. J Protozool 11: 386-394 Keeling PJ (2002) Molecular phylogenetic position of Trichomitopsis termopsidis (Parabasalia) and evidence for the Trichomitopsiinae. Eur J Protistol 38: 279-286

Research paper thumbnail of New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas

Molecular Phylogenetics and Evolution, 2006

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from... more We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mammalian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and α-tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus Trichomonas.

Research paper thumbnail of Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins

The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate... more The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H2 ,C O 2 and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic

Research paper thumbnail of Retortamonad flagellates are closely related to diplomonads--implications for the history of mitochondrial function in eukaryote evolution

Molecular biology and evolution, 2002

We present the first molecular phylogenetic examination of the evolutionary position of retortamo... more We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weight...

Research paper thumbnail of New evolutionary lineages, unexpected diversity, and host speciWcity in the parabasalid genus Tetratrichomonas

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from... more We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mamma- lian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and -tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus

Research paper thumbnail of Cryptic species within the Tetratrichomonas gallinarum species complex revealed by molecular polymorphism

Fuel and Energy Abstracts, 2005

Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds... more Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds. The pathogenicity of this species is controversial, presenting an unsettled problem as yet. We analysed the polymorphism and genetic relationship among 29 isolates of T. gallinarum obtained from eight bird species and five T. gallinarum-like isolates from the oral cavity and lower respiratory tract of human patients. Two methods were used for the analyses: RAPD and sequencing of 16S rRNA, 5.8S rRNA, ITS1 and ITS2 genes, both producing consistent and well-supported results. The isolates were divided into five groups, A–E, with eleven subgroups. The distance between groups E, D and the cluster A–B–C considerably exceeded usual intraspecific polymorphism seen in trichomonads. Moreover, the largest subgroup, A2 (containing 18 isolates), was divided into three branches according to the host specificity. All isolates from humans were placed into avian subgroups A2 and B2. We conclude that ou...

Research paper thumbnail of Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization

Veterinary Parasitology, 2010

Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary ... more Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.

Research paper thumbnail of Molecular Phytogeny of Parabasalids Based on Small Subunit rRNA Sequences, with Emphasis on the Trichomonadinae Subfamily

The Journal of Eukaryotic Microbiology, 2000

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus h... more We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus hatrachorum strain R 105, Trtratrichonzonas gallinarum, and Pentatrichomonas horninis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The-Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trickomonas species as supported by morphological data, with P. horninis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. hatrackorum within the Trichomonadinae. Trickomitus butrackorum (strains R10S and BUB) and Hypotrichornonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. hatrachorum precedes the Trichomonadinae-Tritrichornonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympku, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrickomonus ,fiptu.s. Monocercomonas sp., Dientamoeha ,fragilis, and the unidentified Reticulitermes javipes gut symbiont 1 remain unclear.

Research paper thumbnail of Genetic Analysis of Giardia from Hoofed Farm Animals Reveals Artiodactyl-Specific and Potentially Zoonotic Genotypes

Journal of Eukaryotic Microbiology, 2007

Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture o... more Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A-one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from humanderived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group I1 G. inresrinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.